RS7 antibodies

ABSTRACT

This invention relates to monovalent and multivalent, monospecific binding proteins and to multivalent, multispecific binding proteins. One embodiment of these binding proteins has one or more binding sites where each binding site binds with a target antigen or an epitope on a target antigen. Another embodiment of these binding proteins has two or more binding sites where each binding site has affinity towards different epitopes on a target antigen or has affinity towards either a target antigen or a hapten. The present invention further relates to recombinant vectors useful for the expression of these functional binding proteins in a host. More specifically, the present invention relates to the tumor-associated antigen binding protein designated RS7, and other EGP-1 binding-proteins. The invention further relates to humanized, human and chimeric RS7 antigen binding proteins, and the use of such binding proteins in diagnosis and therapy.

[0001] This application claims priority to U.S. Provisional ApplicationNo. 60/360,299, which is incorporated herein by reference in itsentirety.

FIELD OF THE INVENTION

[0002] This invention relates to monovalent and multivalent,monospecific binding proteins and to multivalent, multispecific bindingproteins. One embodiment of these binding proteins has one or morebinding sites where each binding site binds with a target antigen or anepitope on a target antigen. Another embodiment of these bindingproteins has two or more binding sites where each binding site hasaffinity towards different epitopes on a target antigen or has affinitytowards either a target antigen or a hapten. The present inventionfurther relates to recombinant vectors useful for the expression ofthese functional binding proteins in a host. More specifically, thepresent invention relates to the tumor-associated antigen bindingprotein designated RS7. The invention further relates to humanized RS7antigen binding proteins, and the use of such binding proteins indiagnosis and therapy.

BACKGROUND OF THE INVENTION

[0003] Man-made binding proteins, in particular monoclonal antibodiesand engineered antibodies or antibody fragments, have been tested widelyand shown to be of value in detection and treatment of various humandisorders, including cancers, autoimmune diseases, infectious diseases,inflammatory diseases, and cardiovascular diseases (Filpula and McGuire,Exp. Opin. Ther. Patents (1999) 9: 231-245). For example, antibodieslabeled with radioactive isotopes have been tested to visualize tumorsafter injection to a patient using detectors available in the art. Theclinical utility of an antibody or an antibody-derived agent isprimarily dependent on its ability to bind to a specific targetedantigen. Selectivity is valuable for delivering a diagnostic ortherapeutic agent, such as isotopes, drugs, toxins, cytokines, hormones,growth factors, enzymes, conjugates, radionuclides, or metals, to atarget location during the detection and treatment phases of a humandisorder, particularly if the diagnostic or therapeutic agent is toxicto normal tissue in the body.

[0004] The potential limitations of antibody systems are discussed inGoldenberg, The American Journal of Medicine (1993) 94: 298-299. Theimportant parameters in the detection and treatment techniques are theamount of the injected dose specifically localized at the site(s) wheretarget cells are present and the uptake ratio, i.e. the ratio of theconcentration of specifically bound antibody to that of theradioactivity present in surrounding normal tissues. When an antibody isinjected into the blood stream, it passes through a number ofcompartments as it is metabolized and excreted. The antibody must beable to locate and bind to the target cell antigen while passing throughthe rest of the body. Factors that control antigen targeting includelocation, size, antigen density, antigen accessibility, cellularcomposition of pathologic tissue, and the pharmacokinetics of thetargeting antibodies. Other factors that specifically affect tumortargeting by antibodies include expression of the target antigens, bothin tumor and other tissues, and bone marrow toxicity resulting from theslow blood-clearance of the radiolabeled antibodies. The amount oftargeting antibodies accreted by the targeted tumor cells is influencedby the vascularization and barriers to antibody penetration of tumors,as well as intratumoral pressure. Non-specific uptake by non-targetorgans such as the liver, kidneys or bone-marrow is another potentiallimitation of the technique, especially for radioimmunotherapy, whereirradiation of the bone marrow often causes the dose-limiting toxicity.

[0005] One suggested approach, referred to as direct targeting, is atechnique designed to target specific antigens with antibodies carryingdiagnostic or therapeutic radioisotopes. In the context of tumors, thedirect targeting approach utilizes a radiolabeled anti-tumormonospecific antibody that recognizes the target tumor through itsantigens. The technique involves injecting the labeled monospecificantibody into the patient and allowing the antibody to localize at thetarget tumor to obtain diagnostic or therapeutic benefits. The unboundantibody clears the body. This approach can be used to diagnose or treatadditional mammalian disorders.

[0006] Another suggested solution, referred to as the “AffinityEnhancement System” (AES), is a technique especially designed toovercome deficiencies of tumor targeting by antibodies carryingdiagnostic or therapeutic radioisotopes (U.S. Pat. No. 5,256,395 (1993),Barbet et al., Cancer Biotherapy & Radiopharmaceuticals (1999) 14:153-166). The AES utilizes a radiolabeled hapten and ananti-tumor/anti-hapten bispecific binding protein that recognizes boththe target tumor and the radioactive hapten. Haptens with higher valencyand binding proteins with higher specificity may also be utilized forthis procedure. The technique involves injecting the binding proteininto the patient and allowing it to localize at the target tumor. Aftera sufficient amount of time for the unbound binding protein to clearfrom the blood stream, the radiolabeled hapten is administered. Thehapten binds to the antibody-antigen complex located at the site of thetarget cell to obtain diagnostic or therapeutic benefits. The unboundhapten clears the body. Barbet mentions the possibility that a bivalenthapten may crosslink with a bispecific antibody, when the latter isbound to the tumor surface. As a result, the radiolabeled complex ismore stable and stays at the tumor for a longer period of time. Thissystem can be used to diagnose or treat mammalian disorders.

[0007] There remains a need in the art for production of multivalent,monospecific binding proteins that are useful in a direct targetingsystem and for production of multivalent, multispecific binding proteinsthat are useful in an affinity enhancement system. Specifically, thereremains a need for a binding protein that exhibits enhanced uptake attargeted antigens, decreased concentration in the blood, and optimalprotection of normal tissues and cells from toxic pharmaceuticals.

SUMMARY OF THE INVENTION

[0008] Accordingly, it is an object of the present invention to providea monospecific monoclonal antibody and fragments thereof that recognizesa tumor-associated antigen, defined as epithelial glycoprotein-1 (EGP-1)by the murine MAb RS7-3G11 raised against human non-small-cell lungcarcinoma. The RS7 antigen has been designated as EGP-1 (epithelialglycoprotein-1) following the proposal of the 3^(rd) International IASLCWorkshop on Lung Tumor and Differentiation Antigens. At least oneepitope associated with EGP-1 is alternatively referred to as TROP2 inthe literature. In a preferred embodiment, the antibody or antibodyfragment of the present invention binds the same epitope as the murineRS7 antibody disclosed by Stein (infra) and other earlier studies.Alternatively, the antibody or fragment may bind an epitope distinctfrom the epitope that the murine RS7 antibody disclosed by Stein binds.In a preferred embodiment, the anti-EGP-1, or anti-TROP2 antibody orfragment thereof is a chimeric, humanized, or fully human RS7 antibodyor fragment thereof.

[0009] For example, contemplated in the present invention is a humanizedantibody or fragment thereof, wherein the complementarity determiningregions (CDRs) of the light chain variable region of the humanized RS7MAb comprises CDR1 comprising an amino acid sequence of KASQDVSIAVA;CDR2 comprising an amino acid sequence of SASYRYT; and CDR3 comprisingan amino acid sequence of QQHYITPLT. Another embodiment of the presentinvention is a humanized antibody or fragment thereof, wherein the CDRsof the heavy chain variable region of the humanized RS7 MAb comprisesCDR1 comprising an amino acid sequence of NYGMN; CDR2 comprising anamino acid sequence of WINTYTGEPTYTDDFKG and CDR3 comprising an aminoacid sequence of GGFGSSYWYFDV. Also preferred, the humanized antibody orfragment thereof of comprises the CDRs of a murine RS7 MAb and theframework region (FR) of the light and heavy chain variable regions of ahuman antibody, wherein the CDRs of the light chain variable region ofthe humanized RS7 MAb comprises CDR1 comprising an amino acid sequenceof KASQDVSIAVA; CDR2 comprising an amino acid sequence of SASYRYT; andCDR3 comprising an amino acid sequence of QQHYITPLT; and the CDRs of theheavy chain variable region of the humanized RS7 MAb comprises CDR1comprising an amino acid sequence of NYGMN; CDR2 comprising an aminoacid sequence of WINTYTGEPTYTDDFKG and CDR3 comprising an amino acidsequence of GGFGSSYWYFDV. Still preferred, the humanized antibody orfragment thereof further comprises the FRs of the light and heavy chainconstant regions of a human antibody.

[0010] In a preferred embodiment, the humanized RS7 antibody or fragmentcomprises a FR of a light and/or heavy chain that comprises at least oneamino acid substituted by an amino acid residue found at a correspondinglocation in the RS7 murine antibody. For example, at least one of thesubstituted amino acids is preferably at a location selected from thegroup consisting of residue 38, 46, 68 and 91 of the murine heavy chainvariable region of FIG. 3B, and/or at least one of the substituted aminoacids is preferably at a location selected from the group consisting ofresidue 20, 85 and 100 of the murine light chain variable region of FIG.3A.

[0011] Also described in the present invention is an antibody fusionprotein or fragment thereof that comprises at least two anti-EGP-1 MAbor fragments thereof, wherein the MAb or fragments thereof are selectedfrom the anti-EGP-1 MAb or fragments thereof of the present invention.In a related vein, the antibody fusion protein or fragment thereofcomprises at least one first anti-EGP-1 MAb or fragment thereof of anyof the anti-EGP-1 antibodies of the present invention and at least onesecond MAb or fragment thereof, other than the anti-EGP antibodies orfragment thereof in the present invention. For example, the secondantibody or fragment thereof may be a carcinoma-associated antibody orfragment thereof. Another preferred embodiment is a fusion protein orfragment thereof that comprises two different epitope-binding anti-EGP-1antibodies or fragments thereof.

[0012] It is one object of this invention to provide a multispecificantibody and fragments thereof that recognize more than one epitope onthe RS7 antigen or that has affinity for the RS7 antigen and for ahapten molecule. The latter binding protein is useful for pretargeting atarget antigen. Accordingly, a method of delivering a diagnostic agent,a therapeutic agent, or a combination thereof to a target, comprising:(i) administering to a subject a multivalent, multispecific MAb, orfragment thereof (ii) waiting a sufficient amount of time for an amountof the non-binding protein to clear the subject's blood stream; and(iii) administering to said subject a carrier molecule comprising adiagnostic agent, a therapeutic agent, or a combination thereof, thatbinds to a binding site of said antibody, is also described.

[0013] It is a further object of this invention to provide a method ofdelivering a diagnostic or therapeutic agent to a targeted disease thatexpresses EGP-1 antigen. For example, a method of delivering adiagnostic or therapeutic agent, or a combination thereof, to a targetcomprising (i) providing a composition that comprises an anti-EGP-1antibody or fragment thereof bound to at least one thereapeutic and/ordiagnostic agent and (ii) administering to a subject in need thereofsaid composition, is described. Preferably, the diagnostic ortherapeutic agent is selected from the group consisting of an isotope,drug, toxin, imuno, modulator, hormone, enzyme, growth factor,radionuclide, metal, contrast agent, and detecting agent.

[0014] In another embodiment of the present invention, the method fordelivering a diagnostic agent, a therapeutic agent, or a combinationthereof to a target comprises (i) administering to a subject amultivalent, multispecific antibody or fragment comprising one or moreantigen-binding sites having affinity toward an EGP-1 target antigen andone or more hapten binding sites having an affinity toward a haptenmolecule, (ii) waiting a sufficient amount of time for an amount of thenon-binding antibody or fragment to clear a subject's blood stream, and(iii) administering to said subject a hapten comprising a diagnosticagent, a therapeutic agent, or a combination thereof.

[0015] Another object of the present invention to provide a cancer celltargeting diagnostic or therapeutic conjugate that comprises ananti-EGP-1 MAb or fragment thereof or an antibody fusion protein orfragment thereof of any one of antibodies of the present invention andwherein the anti-EGP-1 antibody or fragment thereof is bound to at leastone diagnostic or therapeutic agent. A suitable therapeutic agent is adrug that possesses the pharmaceutical property selected from the groupconsisting of an antimitotic, alkylating, antimetabolite,antiangiogenic, apoptotic, alkyloid antibiotic, and combinationsthereof. Also preferred is a therapeutic agent selected from the groupconsisting of a nitrogen mustard, ethylenimine derivative, alkylsulfonate, nitrosurea, traizene, folic acid analog, antracycline,taxane, COX-2 inhibitor, tyrosine kinase inhibitor, pyrimidine analog,purine analog, antibiotic, enzyme, epipodophyllotoxin, platinumcoordination complex, vinca alkaloid, substituted urea, methyl hydrazinederivative, adrenocorticol suppressant, antagonist, endostatin taxol,camptothecins, doxorubicin, doxorubicin analog, and a combinationthereof. Preferably, the diagnostic agent is selected from the groupconsisting of a photoactive radionuclide, preferably between 25 and 4000keV, and a contrast agent.

[0016] In a preferred embodiment, a DNA sequence comprising a nucleicacid encoding a MAb or fragment that contains a anti-EGP-1 MAb orfragment thereof of the present invention; an antibody fusion protein orfragment thereof containing at least two of said MAbs or fragmentsthereof; an antibody fusion protein or fragment thereof containing atleast one first anti-EGP-1 MAb or fragment thereof containing the MAb orfragment thereof of the anti-EGP-1 antibodies and fragments of thepresent invention and at least one second MAb or fragment thereof, otherthan the anti-EGP-1 MAb or fragment thereof described herein; or anantibody fusion protein or fragment thereof comprising at least onefirst MAb or fragment thereof comprising said MAb or fragment thereof ofany of the antibodies described herein and at least one second MAb orfragment thereof, other than the MAb or fragment thereof of any one ofthe antibodies described herein, wherein the second MAb is reactive withan antigen selected from the group consisting of EGP-2, MUC 1-4, A33,CSAp, CEA, Le(y), Tn, Tag-72, PSMA, PSA, EGFR, HER2/neu, AFP, HCG,HCG-beta, ferritin, PAP, PLAP, EGP-2, histone, cytokeratin, Tenascin,CanAg, kidney cancer G 250, VGFR1, VGFR2, PAM4-antigen, oncogeneproducts, or a combination thereof. The second MAb may instead bereactive with vascular endothelial antigens associated with tumors, suchas VEGF (vascular endothelial growth factor) and P1GF (placenta growthfactor). Selection of the second antibody is dependent on tumor celltype. For example, anti-PSMA or anti-PSA antibodies may be used fortreating or diagnosing prostate cancer, anti-CEA or anti-MUC1, MUC2,MUC3 and MUC4 antibodies for breast, ovarian, lung, and colon cancer,EGFR for colon and head and neck cancers, anti-CSAp antibodies for colonand ovarian cancer, and anti-HER/neu for breast, ovarian and othercancers. These are merely given as examples, and are not intended to belimiting. Expression vectors and host cells containing this DNA sequenceare also preferred embodiments of the present invention.

[0017] Also provided herein are methods for diagnosing and treating amalignancy. For example, a method for diagnosing or treating cancer,comprises (i) administering to a subject in need thereof a multivalent,multispecific antibody or fragment comprising one or moreantigen-binding sites having affinity toward an EGP-1 target antigen andone or more hapten binding sites having an affinity toward a haptenmolecule; (ii) waiting a sufficient amount of time for an amount of thenon-binding protein to clear the subject's blood stream; and (iii)administering to said subject a hapten comprising a diagnostic agent, atherapeutic agent, or a combination thereof, that binds to a bindingsite of said antibody.

[0018] Likewise, the methods for diagnosing and treating a malignancymay comprise administering a therapeutically effective amount of ananti-EGP-1 fusion protein or fragment thereof or a therapeutic conjugatecomprising a EGP-1 MAb or fragment thereof, wherein the EGP-1 MAb orfragment thereof or antibody fusion protein or fragment thereof is boundto at least one therapeutic agent in a pharmaceutically suitableexcipient. In a related vein, naked anti-EGP-1 antibodies and fragmentsthereof, including naked anti-EGP-1 fusion proteins and fragmentsthereof, can also be used for treating a malignancy. Naked anti-EGP-1antibodies may be used for in vitro diagnosis of a malignancy, forexample with immunoassays or immunohistochemistry, but not for in vivodiagnosis, unless this involves a pretargeting technology, such as AES.Labeled EGP-1 antibodies, however, may be used for in vivo diagnosis andtreatment of a malignancy. For example, described herein is a method oftreating a cancer cell in a subject comprising (i) administering to asubject a therapeutically effective amount of a composition containingan anti-EGP-1 MAb or fragment thereof or an antibody fusion protein orfragment thereof (ii) formulating the EGP-1 MAb or fragment thereof orantibody fusion protein or fragment thereof in a pharmaceuticallysuitable excipient. Similarly, combinations of naked MAbs and fragmentsthereof with conjugated MAbs or fragments thereof or fusion proteins orfragments thereof for diagnosis and treatment are also contemplated inthe instant invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0019]FIG. 1 shows a comparison of mRS7, cAb-Vκ#23 (cRS7), and cAb-Vκ#1in competitive binding assays. Varying concentrations of competing Abswere used to compete with the binding of a constant amount ofbiotinylated mRS7 antibody. Results indicate that the Vκ#1 light chaindoes not bind the RS7 antigen.

[0020]FIG. 2 shows the DNA and amino acid sequences encoding (A) RS7 Vκcloned by 5′ RACE and (B) RS7 VH cloned by RT-PCR. The putative CDRregions are underlined and indicated. Nucleotide residues are numberedsequentially. Kabat's Ig molecule numbering is used for amino acidresidues. In (B), the numbering for the residues with a letter (on top)is the number of preceding residues plus the letter, e.g., the numberfor T following N52 is 52A; the numbers for N, N and L following 182 are82A, 82B and 82C, respectively.

[0021]FIG. 3 shows the amino acid sequence alignment of (A) humanSA-1A′cl, murine RS7, and hRS7 V_(K) chains and (B) human RF-TS3, murineRS7, and hRS7 V_(H) chains. In (A), dots indicate the residues in RS7are identical to the corresponding residues in SA-1A′cl. Dashesrepresent gaps introduced to aid the alignment. Boxed represent the CDRregions. Both N- and C-terminal residues (underlined) of hRS7 are fixedby the staging vector used. Therefore, the corresponding terminalresidues of RS7 are not compared with that of the human sequence.Kabat's numbering scheme is used. In (B), dots indicate the residues inRS7 are identical to the corresponding residues in RF-TS3. Dashesrepresent gaps introduced to aid the alignment. Boxed represent the CDRregions. Both N- and C-terminal residues (underlined) of hRS7 are fixedby the staging vector used. Therefore, the corresponding terminalresidues of RS7 are not compared with that of the human VH sequence.

[0022]FIG. 4 shows the DNA and amino acid sequences for (A) humanizedRS7 V_(K) and (B) humanized RS7 V_(H). The bold and underlined sectionsof the amino acid sequences indicate the CDRs as defined by the Kabatnumbering scheme.

[0023]FIG. 5 shows the (A) light chain cDNA and amino acid sequences forhumanized RS7 Vκ and (B) heavy chain cDNA and amino acid sequences forhumanized RS7 V_(H). The underlined sections of the amino acid sequencesindicate the leader peptide sequence for secretion. “*” indicates thestop codon.

[0024]FIG. 6 shows a comparison of mRS7, cRS7, and hRS7 in competitivebinding assays. Varying concentrations of competing Abs were used tocompete with the binding of a constant amount of Biotinylated RS7 to theAg coated in 96-well ELISA plates. hRS7 showed comparable blockingactivity as that of RS7 and cRS7.

[0025]FIG. 7 shows the light chain cDNA and amino acid sequences forhumanized RS7 V_(K) . The underlined sections of the amino acidsequences indicate the leader peptide sequence for secretion. “*”indicates the stop codon. The lysine residues are also underlined.

[0026]FIG. 8 shows the heavy chain cDNA and amino acid sequences forhumanized RS7 V_(K). The underlined sections of the amino acid sequencesindicate the leader peptide sequence for secretion. “*” indicates thestop codon. The lysine residues are also underlined.

[0027]FIG. 9 indicates the structure of the residualizing moietiesIMP-R4, IMP-R5 and IMP-R8.

[0028]FIG. 10 is a bar graph of dosimetry due to radioiodinated hRS7 inthe MDA-MB-468 tumor model.

[0029]FIG. 11 provides a series of graphs demonstrating the effects ofradioimmunotherapy on tumor growth of breast cancer xenografts in nudemice.

[0030]FIG. 12 is a series of graphs evaluating toxicity afterradioimmunotherapy treatment of breast cancer xenografts in nude mice.

[0031]FIG. 13 is a graph demonstrating relative mean tumor volumes(MTV).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0032] Unless otherwise specified, “a” or “an” means “one or more.”

[0033] An RS7 antibody (previously designated RS7-3G11) is a murine IgG₁raised against a crude membrane preparation of a human primary squamouscell carcinoma from the lung. See Stein et al., Cancer Res. 50: 1330(1990), which is fully incorporated by reference. The RS7 antibodyrecognizes a tumor-associated antigen, which was defined by the murineMAb RS7-3G11 raised against human non-small-cell lung carcinoma. Steinet al discloses that the RS7 antibody recognizes a 46-48 kDaglycoprotein, characterized as cluster 13. Stein et al., Int. J. CancerSupp. 8:98-102 (1994). See also, Basu et al., Int. J. Cancer 52:472-479(1995). The antigen has been designated as EGP-1 (epithelialglycoprotein-1) following the proposal of the 3^(rd) International IASLCWorkshop on Lung Tumor and Differentiation Antigens. See, for exampleDeLeij et al., Int. J. Cancer Supp., 8:60-63-(1994). Accordingly, asdescribed herein, the RS7 and EGP-1 antigens are synonymous. The EGP-1antigen is also referred to as TROP2 in the literature, but there may bemultiple epitopes of both EGP-1 and TROP2.

[0034] Flow cytometry and immunohistochemical staining studies haveshown that the RS7 MAb detects antigen on a variety of tumor types, withlimited binding to normal human tissue. (Stein et al., (1990), supra).The RS7 antibody is reactive with an EGP-1 glycoprotein, which can berapidly internalized. EGP-1 is expressed primarily by carcinomas such ascarcinomas of the lung, stomach, urinary bladder, breast, ovary, uterus,and prostate. Localization and therapy studies using radiolabeled murineRS7 MAb in animal models have demonstrated tumor targeting andtherapeutic efficacy (Stein et al., (1990), supra. Stein et al., (1991),supra).

[0035] A more recent study has demonstrated strong RS7 staining intumors from the lung, breast, bladder, ovary, uterus, stomach, andprostate. See Stein et al., Int. J. Cancer 55: 938 (1993), which isfully incorporated by reference. Moreover, the lung cancer cases in thisstudy comprised both squamous cell carcinomas and adenocarcinomas. Id.Both cell types stained strongly, indicating that the RS7 antibody doesnot distinguish between histologic classes of non-small-cell carcinomaof the lung.

[0036] As discussed supra, the RS7 MAb is rapidly internalized intotarget cells (Stein et al. (1993), supra). The internalization rateconstant for RS7 MAb is intermediate between the internalization rateconstants of two other rapidly internalizing MAbs, which have beendemonstrated to be useful for immunotoxin production. Id. It is welldocumented that the internalization of immunotoxin conjugates is anabsolute requirement for anti-tumor activity. (Pastan et al., Cell47:641 (1986)). Internalization of drug immunoconjugates also has beendescribed as a major factor in anti-tumor efficacy. (Yang et al., Proc.Nat'l Acad. Sci. USA 85: 1189 (1988)). Therefore, the RS7 antigen may bean important target for those types of immunotherapy that requireinternalization of the therapeutic agent.

[0037] Thus, studies with the RS7 MAb indicate that the antibodyexhibits several important properties, which make it a candidate forclinical diagnostic and therapeutic applications. Since the RS7 antigenprovides a useful target for diagnosis and therapy, it is desirable toobtain a MAb that recognizes an epitope of the RS7 antigen. Moreover,the availability of chimeric, humanized and human RS7 antibodies isessential for the development of a double-determinant enzyme-linkedimmunosorbent assay (ELISA), which is desireable for detecting the RS7antigen in clinical samples, and essential for in vivo applications inhumans.

[0038] To this end, the present invention describes chimeric, humanizedand human antibodies and fragments thereof that bind the RS7 antigen andcan be used for diagnostic and therapeutic methods. Humanized antibodiesand antibody fragments are described in Provisional U.S. Applicationtitled “Anti-CD20 Antibodies And Fusion Proteins Thereof And Methods OfUse”, Attorney Docket No. 18733/1073, U.S. Provisional No. 60/356,132,U.S. Provisional Application No. 60/416,232 and Attorney Docket No.18733/1155; hMN-14 antibodies, such as those disclosed in U.S.application Ser. No. 5,874,540, which is a Class IIIanti-carcinoembryonic antigen antibody (anti-CEA antibody); Mu-9antibodies, such as those described in U.S. application Ser. No.10/116,116; AFP antibodies, such as those described in U.S. ProvisionalApplication No. 60/399,707; PAM4 antibodies, such as those described inProvisional U.S. Application titled “Monoclonal Antibody cPAM4”,Attorney Docket No. 18733/1102; RS7 antibodies, such as those describedin U.S. Provisional Application No. 60/360,229 ; and CD22 antibodies,such as those disclosed in U.S. Pat. Nos. 5,789,554 and 6,187,287 andU.S. application Ser. Nos. 09/741,843 and 09/988,013, all of which areincorporated herein by reference in their entirety. A chimeric antibodyas disclosed herein is a recombinant protein that contains the variabledomains including the complementarity determining regions (CDRs) of anantibody derived from one species, preferably a rodent antibody, whilethe constant domains of the antibody molecule is derived from those of ahuman antibody. For veterinary applications, the constant domains of thechimeric antibody may be derived from that of other species. A humanizedantibody is a recombinant protein in which the CDRs from an antibody ofone species, e.g., a rodent antibody, are transferred from the heavy andvariable chains of the rodent antibody into human heavy and lightvariable domains.

[0039] In a preferred embodiment, the RS7 antibody is humanized. Becausenon-human monoclonal antibodies can be recognized by the human host as aforeign protein, and repeated injections can lead to harmfulhypersensitivity reactions, humanization of a murine RS7 sequences canreduce the adverse immune response that patients may experience. Formurine-based monoclonal antibodies, this is often referred to as a HumanAnti-Mouse Antibody (HAMA) response. Another embodiment of the presentinvention is an anti-EGF-1 antibody or fragment thereof that is asubhuman primate anti-EGP-1 antibody, murine monoclonal anti-EGP-1antibody (restricted to veterinary applications), chimeric anti-EGP-1antibody, human anti-EGP-1 antibody, and humanized anti-EGP-1 antibody.Preferably, the chimeric, human and humanized anti-EGP-1 antibodycomprises constant and hinge regions of a human IgG1. Also preferred,some human residues in the framework regions of the humanized RS7antibody or fragments thereof are replaced by their murine counterparts.It is also preferred that a combination of framework sequences from 2different human antibodies are used for V_(H). The constant domains ofthe antibody molecule are derived from those of a human antibody.

[0040] Another preferred embodiment of the present invention is a humanRS7 antibody. A human antibody is an antibody obtained from transgenicmice that have been “engineered” to produce specific human antibodies inresponse to antigenic challenge. In this technique, elements of thehuman heavy and light chain locus are introduced into strains of micederived from embryonic stem cell lines that contain targeted disruptionsof the endogenous heavy chain and light chain loci. The transgenic micecan synthesize human antibodies specific for human antigens, and themice can be used to produce human antibody-secreting hybridomas. Methodsfor obtaining human antibodies from transgenic mice are described byGreen et al., Nature Genet. 7:13 (1994), Lonberg et al., Nature 368:856(1994), and Taylor et al., Int. Immun. 6:579 (1994). A fully humanantibody also can be constructed by genetic or chromosomal transfectionmethods, as well as phage display technology, all of which are known inthe art. See for example, McCafferty et al., Nature 348:552-553 (1990)for the production of human antibodies and fragments thereof in vitro,from immunoglobulin variable domain gene repertoires from unimmunizeddonors. In this technique, antibody variable domain genes are clonedin-frame into either a major or minor coat protein gene of a filamentousbacteriophage, and displayed as functional antibody fragments on thesurface of the phage particle. Because the filamentous particle containsa single-stranded DNA copy of the phage genome, selections based on thefunctional properties of the antibody also result in selection of thegene encoding the antibody exhibiting those properties. In this way, thephage mimics some of the properties of the B cell. Phage display can beperformed in a variety of formats, for their review, see e.g. Johnsonand Chiswell, Current Opiniion in Structural Biology 3:5564-571 (1993).

[0041] The antibody and fragments thereof of the present invention ispreferably raised against a crude membrane preparation from a humanprimary squamous cell carcinoma of the lung. Also preferred, the RS7antibody and fragments thereof is raised against a membrane preparationof viable cells from a human ovarian carcinoma cell line. Stillpreferred, the RS7 antigen is provided by viable Colo 316 cells. In arelated vein, the RS7 antibody can be obtained using a substantiallypure preparation of the RS7 antigen. A substantially pure protein is aprotein that is essentially free from contaminating cellular components,which are associated with the protein in nature. As described herein,the term “RS7 antibody” also includes chimeric, human and humanized RS7antibodies.

[0042] Preparation of Chimeric, Humanized and Human RS7 Antibodies

[0043] Monoclonal antibodies to specific antigens may be obtained bymethods known to those skilled in the art. See, for example, Kohler andMilstein, Nature 256: 495 (1975), and Coligan et al. (eds.), CURRENTPROTOCOLS IN IMMUNOLOGY, VOL. 1, pages 2.5.1-2.6.7 (John Wiley & Sons1991) (hereinafter “Coligan”). Briefly, RS7 antigen MAbs, such as RS7,can be obtained by injecting mice with a composition comprising the RS7antigen, verifying the presence of antibody production by removing aserum sample, removing the spleen to obtain B-lymphocytes, fusing theB-lymphocytes with myeloma cells to produce hybridomas, cloning thehybridomas, selecting positive clones which produce antibodies to RS7antigen, culturing the clones that produce antibodies to RS7 antigen,and isolating RS7 antibodies from the hybridoma cultures.

[0044] After the initial raising of antibodies to the immunogen, theantibodies can be sequenced and subsequently prepared by recombinanttechniques. Humanization and chimerization of murine antibodies andantibody fragments are well known to those skilled in the art. Forexample, humanized monoclonal antibodies are produced by transferringmouse complementary determining regions from heavy and light variablechains of the mouse immunoglobulin into a human variable domain, andthen, substituting human residues in the framework regions of the murinecounterparts. The use of antibody components derived from humanizedmonoclonal antibodies obviates potential problems associated with theimmunogenicity of murine constant regions.

[0045] A human antibody of the present invention, i.e., human EGP-1 MAbsor other human antibodies, such as anti-EGP-2, MUC1-4, CEA, CC49, CSAp,PSMA, PSA, EGFR, A33 and HER2/neu MAbs for combination therapy withhumanized, chimeric or human RS7 antibodies, can be obtained from atransgenic non-human animal. See, e.g., Mendez et al., Nature Genetics,15: 146-156 (1997); U.S. Pat. No. 5,633,425, which are incorporated intheir entirety by reference. A human antibody of the present inventionthat can be used for combination therapy may also be reactive with anantigen selected from the group consisting of Le(y), Tn, Tag-72, AFP,HCG, HCG-beta, ferritin, PAP, EGP-2, histone, cytokeratin, Tenascin,CanAg, kidney cancer G 250, VGFR1, VGFR2, or a combination thereof Forexample, a human antibody can be recovered from a transgenic mousepossessing human immunoglobulin loci. The mouse humoral immune system ishumanized by inactivating the endogenous immunoglobulin genes andintroducing human immunoglobulin loci. The human immunoglobulin loci areexceedingly complex and comprise a large number of discrete segmentswhich together occupy almost 0.2% of the human genome. To ensure thattransgenic mice are capable of producing adequate repertoires ofantibodies, large portions of human heavy- and light-chain loci must beintroduced into the mouse genome. This is accomplished in a stepwiseprocess beginning with the formation of yeast artificial chromosomes(YACs) containing either human heavy- or light-chain immunoglobulin lociin germline configuration. Since each insert is approximately 1 Mb insize, YAC construction requires homologous recombination of overlappingfragments of the immunoglobulin loci. The two YACs, one containing theheavy-chain loci and one containing the light-chain loci, are introducedseparately into mice via fusion of YAC-containing yeast spheroblastswith mouse embryonic stem cells. Embryonic stem cell clones are thenmicroinjected into mouse blastocysts. Resulting chimeric males arescreened for their ability to transmit the YAC through their germlineand are bred with mice deficient in murine antibody production. Breedingthe two transgenic strains, one containing the human heavy-chain lociand the other containing the human light-chain loci, creates progeny,which produce human antibodies in response to immunization.

[0046] General techniques for cloning murine immunoglobulin variabledomains are described, for example, by the publication of Orlandi etal., Proc. Nat'l Acad. Sci. USA 86: 3833 (1989), which is incorporatedby reference in its entirety. Techniques for producing humanized MAbsare described, for example, by Carter et al., Proc. Nat'l Acad. Sci. USA89: 4285 (1992), Singer et al., J. Immun. 150: 2844 (1992), Mountain etal. Biotechnol. Genet. Eng. Rev. 10: 1 (1992), and Coligan at pages10.19.1-10.19.11, each of which is hereby incorporated by reference.

[0047] In general, the Vκ (variable light chain) and V_(H) (variableheavy chain) sequences for RS7 antibodies can be obtained by a varietyof molecular cloning procedures, such as RT-PCR, 5′-RACE, and cDNAlibrary screening. Specifically, the VH and Vκ genes of the MAb RS7 werecloned by PCR amplification from the hybridoma cells by RT-PCR and 5′RACE, respectively, and their sequences determined by DNA sequencing. Toconfirm their authenticity, the cloned V_(L) and V_(H) genes can beexpressed in cell culture as a chimeric Ab as described by Orlandi etal., (Proc. Natl. Acad. Sci., USA, 86: 3833 (1989)) which isincorporated by reference. Based on the V gene sequences, a humanizedRS7 antibody can then be designed and constructed as described by Leunget al. (Mol. Immunol., 32: 1413 (1995)), which is incorporated byreference. cDNA can be prepared from any known hybridoma line ortransfected cell line producing a murine or chimeric RS7 antibody bygeneral molecular cloning techniques (Sambrook et al., MolecularCloning, A laboratory manual, 2^(nd) Ed (1989)). In a preferredembodiment, the RS7 hybridoma line is used. The Vκ sequence for the mAbmay be amplified using the primers VK1BACK and VK1FOR (Orlandi et al.,1989) or the extended primer set described by Leung et al.(BioTechniques, 15: 286 (1993)), which is incorporated by reference,while V_(H) sequences can be amplified using the primer pairVH1BACK/VH1FOR (Orlandi et al., 1989 above), or the primers annealing tothe constant region of murine IgG described by Leung et al. (Hybridoma,13:469 (1994)), which is incorporated by reference. The PCR reactionmixtures containing 10 μl of the first strand cDNA product, 10 μl of 10×PCR buffer [500 mM KCl, 100 mM Tris-HCl (pH 8.3), 15 mM MgCl₂, and 0.01%(w/v) gelatin] (Perkin Elmer Cetus, Norwalk, Conn.), 250 μM of eachdNTP, 200 nM of the primers, and 5 units of Taq DNA polymerase (PerkinEhner Cetus) can be subjected to 30 cycles of PCR. Each PCR cyclepreferably consists of denaturation at 94° C. for 1 min, annealing at 50° C. for 1.5 min, and polymerization at 72° C. for 1.5 min. Amplified Vκand VH fragments can be purified on 2% agarose (BioRad, Richmond,Calif.). Similarly, the humanized V genes can be constructed by acombination of long oligonucleotide template syntheses and PCRamplification as described by Leung et al. (Mol. Immunol., 32: 1413(1995)).

[0048] PCR products for Vκ can be subcloned into a staging vector, suchas a pBR327-based staging vector, VKpBR, that contains an Ig promoter, asignal peptide sequence and convenient restriction sites to facilitatein-frame ligation of the Vκ PCR products. PCR products for V_(H) can besubcloned into a similar staging vector, such as the pBluescript-basedVHpBS. Individual clones containing the respective PCR products may besequenced by, for example, the method of Sanger et al. (Proc. Natl.Acad. Sci., USA, 74: 5463 (1977)), which is incorporated by reference.

[0049] The DNA sequences described herein are to be taken as includingall alleles, mutants and variants thereof, whether occurring naturallyor induced.

[0050] The expression cassettes containing the Vκ and VH, together withthe promoter and signal peptide sequences can be excised from VKpBR andVHpBS, respectively, by double restriction digestion as HindIII-BamHIfragments. The Vκ and VH expression cassettes can then be ligated intoappropriate expression vectors, such as pKh and pG1g, respectively(Leung et al., Hybridoma, 13:469 (1994)). The expression vectors can beco-transfected into an appropriate cell, e.g., myeloma Sp2/0-Ag14 (ATCC,VA), colonies selected for hygromycin resistance, and supernatant fluidsmonitored for production of a chimeric or humanized RS7 MAb by, forexample, an ELISA assay, as described below. Alternately, the Vκ and VHexpression cassettes can be assembled in the modified staging vectors,VKpBR2 and VHpBS2, excised as XbaI/BamHI and XhoI/BamHI fragments,respectively, and subcloned into a single expression vector, such aspdHL2, as described by Gilles et al. (J. Immunol. Methods 125:191 (1989)and also shown in Losman et al., Cancer, 80:2660 (1997)) for theexpression in Sp2/0-Ag14 cells. Another vector that is useful in thepresent invention is the GS vector, as described in Barnes et al.,Cytotechnology 32:109-123 (2000), which is preferably expressed in theNS0 cell line and CHO cells. Other appropriate mammalian expressionsystems are described in Werner et al., Arzneim.-Forsch./Drug Res.48(II), Nr. 8, 870-880 (1998).

[0051] Co-transfection and assay for antibody secreting clones by ELISA,can be carried out as follows. About 10 μg of VKpKh (light chainexpression vector) and 20 μg of VHpG1g (heavy chain expression vector)can be used for the transfection of 5×10⁶ SP2/0 myeloma cells byelectroporation (BioRad, Richmond, Calif.) according to Co et al., J.Immunol., 148: 1149 (1992) which is incorporated by reference. Followingtransfection, cells may be grown in 96-well microtiter plates incomplete HSFM medium (Life Technologies, Inc., Grand Island, N.Y.) at 37

C., 5% CO₂. The selection process can be initiated after two days by theaddition of hygromycin selection medium (Calbiochem, San Diego, Calif.)at a final concentration of 500 units/ml of hygromycin. Coloniestypically emerge 2-3 weeks post-electroporation. The cultures can thenbe expanded for further analysis.

[0052] Suitable host cells include microbial or mammalian host cells. Apreferred host is the human cell line, PER.C6, which was developed forproduction of MAbs, and other fusion proteins. Accordingly, a preferredembodiment of the present invention is a host cell comprising a DNAsequence encoding and anti-EGP-1 MAb, conjugate, fusion protein orfragments thereof. PER.C6 cells (WO 97/00326) were generated bytransfection of primary human embryonic retina cells, using a plasmidthat contained the Adserotype 5 (Ad5) E1A- and E1B-coding sequences (Ad5nucleotides 459-3510) under the control of the human phosphoglyceratekinase (PGK) promoter. E1A and E1B are adenovirus early gene activationprotein 1A and 1B, respectively. The methods and compositions areparticularly useful for generating stable expression of humanrecombinant proteins of interest that are modified post-translationally,e.g. by glycosylation. Several features make PER.C6 particularly usefulas a host for recombinant protein production, such as PER.C6 is a fullycharacterized human cell line and it was developed in compliance withgood laboratory practices. Moreover, PER.C6 can be grown as a suspensionculture in defined serum-free medium devoid of any human- oranimal-derived proteins and its growth is compatible with rollerbottles, shaker flasks, spinner flasks and bioreactors with doublingtimes of about 35 hrs. Finally, the presence of E1A causes an upregulation of expression of genes that are under the control of the CMVenhancer/promoter and the presence of E13 prevents p53-dependentapoptosis possibly enhanced through over expression of the recombinanttransgene. In one embodiment, the cell is capable of producing 2 to200-fold more recombinant protein and/or proteinaceous substance thanconventional mammalian cell lines.

[0053] Transfectoma clones that are positive for the secretion ofchimeric or humanized heavy chain can be identified by ELISA assay.Briefly, supernatant samples (˜100 μl) from transfectoma cultures areadded in triplicate to ELISA microtiter plates precoated with goatanti-human (GAH)-IgG, F(ab′)₂ fragment-specific antibody (JacksonImmunoResearch, West Grove, Pa.). Plates are incubated for 1 h at roomtemperature. Unbound proteins are removed by washing three times withwash buffer (PBS containing 0.05% polysorbate 20). Horseradishperoxidase (HRP) conjugated GAH-IgG, Fc fragment-specific antibodies(Jackson ImmunoResearch) are added to the wells, (100 μl of antibodystock diluted×10⁴, supplemented with the unconjugated antibody to afinal concentration of 1.0 μg/ml). Following an incubation of 1 h, theplates are washed, typically three times. A reaction solution, [100 μl,containing 167 μg of orthophenylene-diamine (OPD) (Sigma, St. Louis,Mo.), 0.025% hydrogen peroxide in PBS], is added to the wells. Color isallowed to develop in the dark for 30 minutes. The reaction is stoppedby the addition of 50 μl of 4 N HCl solution into each well beforemeasuring absorbance at 490 nm in an automated ELISA reader (Bio-Tekinstruments, Winooski, Vt.). Bound chimeric antibodies are thandetermined relative to an irrelevant chimeric antibody standard(obtainable from Scotgen, Ltd., Edinburg, Scotland).

[0054] Antibodies can be isolated from cell culture media as follows.Transfectoma cultures are adapted to serum-free medium. For productionof chimeric antibody, cells are grown as a 500 ml culture in rollerbottles using HSFM. Cultures are centrifuged and the supernatantfiltered through a 0.2μ membrane. The filtered medium is passed througha protein A column (1×3 cm) at a flow rate of 1 ml/min. The resin isthen washed with about 10 column volumes of PBS and protein A-boundantibody is eluted from the column with 0.1 M glycine buffer (pH 3.5)containing 10 mM EDTA. Fractions of 1.0 ml are collected in tubescontaining 10 μl of 3 M Tris (pH 8.6), and protein concentrationsdetermined from the absorbance at 280/260 nm. Peak fractions are pooled,dialyzed against PBS, and the antibody concentrated, for example, withthe Centricon 30 (Amicon, Beverly, Mass.). The antibody concentration isdetermined by ELISA, as before, and its concentration adjusted to about1 mg/ml using PBS. Sodium azide, 0.01% (w/v), is conveniently added tothe sample as preservative.

[0055] The nucleotide sequences of the primers used to prepare the RS7antibodies are listed in Example 2, below. In a preferred embodiment, ahumanized RS7 antibody or antibody fragment comprises thecomplementarity-determining regions (CDRs) of a murine RS7 MAb and theframework (FR) regions of the light and heavy chain variable regions ofa human antibody and the light and heavy chain constant regions of ahuman antibody, wherein the CDRs of the light chain variable region ofthe humanized RS7 comprises CDR1 comprising an amino acid sequence ofKASQDVSIAVA; CDR2 comprising an amino acid sequence of SASYRYT; and CDR3comprising an amino acid sequence of QQHYITPLT; and the CDRs of theheavy chain variable region of the humanized RS7 MAb comprises CDR1comprising an amino acid sequence of NYGMN; CDR2 comprising an aminoacid sequence of WINTYTGEPTYTDDFKG and CDR3 comprising an amino acidsequence of GGFGSSYWYFDV. Also preferred, the FRs of the light and heavychain variable regions of the humanized antibody comprise at least oneamino acid substituted from said corresponding FRs of the murine RS7MAb.

[0056] RS7 MAbs can be isolated and purified from hybridoma cultures bya variety of well-established techniques. Such isolation techniquesinclude affinity chromatography with Protein-A Sepharose, size-exclusionchromatography, and ion-exchange chromatography. See, for example,Coligan at pages 2.7.1-2.7.12 and pages 2.9.1-2.9.3. Also, see Baines etal., “Purification of Immunoglobulin G (IgG),” in METHODS IN MOLECULARBIOLOGY, VOL. 10, pages 79-104 (The Humana Press, Inc. 1992).

[0057] RS7 MAbs can be characterized by a variety of techniques that arewell-known to those of skill in the art. For example, the ability of anRS7 MAb to bind to the RS7 antigen can be verified using an indirectimmunofluorescence assay, flow cytometry analysis, or Western analysis.

[0058] Production of RS7 Antibody Fragments

[0059] The present invention contemplates the use of fragments of RS7and hRS7 antibodies. Antibody fragments, which recognize specificepitopes, can be generated by known techniques. The antibody fragmentsare antigen binding portions of an antibody, such as F(ab′)₂, Fab′, Fab,Fv, sFv and the like. Other antibody fragments include, but are notlimited to: the F(ab)′₂ fragments which can be produced by pepsindigestion of the antibody molecule and the Fab′ fragments, which can begenerated by reducing disulfide bridges of the F(ab)′₂ fragments. Thesemethods are described, for example, by Goldenberg, U.S. Pat. Nos.4,036,945 and 4,331,647 and references contained therein, which patentsare incorporated herein in their entireties by reference. Also, seeNisonoff et al., Arch Biochem. Biophys. 89: 230 (1960); Porter, Biochem.J. 73: 119 (1959), Edelman et al., in METHODS IN ENZYMOLOGY VOL. 1, page422 (Academic Press 1967), and Coligan at pages 2.8.1-2.8.10 and2.10.-2.10.4. Alternatively, Fab′ expression libraries can beconstructed (Huse et al., 1989, Science, 246:1274-1281) to allow rapidand easy identification of monoclonal Fab′ fragments with the desiredspecificity. The present invention encompasses antibodies and antibodyfragments.

[0060] A single chain Fv molecule (scFv) comprises a VL domain and a VHdomain. The VL and VH domains associate to form a target binding site.These two domains are further covalently linked by a peptide linker (L).A scFv molecule is denoted as either VL-L-VH if the VL domain is theN-terminal part of the scFv molecule, or as VH-L-VL if the VH domain isthe N-terminal part of the scFv molecule. Methods for making scFvmolecules and designing suitable peptide linkers are described in U.S.Pat. No. 4,704,692, U.S. Pat. No. 4,946,778, R. Raag and M. Whitlow,“Single Chain Fvs.” FASEB Vol 9:73-80 (1995) and R. E. Bird and B. W.Walker, “Single Chain Antibody Variable Regions,” TIBTECH, Vol 9:132-137 (1991). These references are incorporated herein by reference.

[0061] An antibody fragment can be prepared by proteolytic hydrolysis ofthe full length antibody or by expression in E. coli or another host ofthe DNA coding for the fragment. An antibody fragment can be obtained bypepsin or papain digestion of full length antibodies by conventionalmethods. For example, an antibody fragment can be produced by enzymaticcleavage of antibodies with pepsin to provide a 5S fragment denotedF(ab′)₂. This fragment can be further cleaved using a thiol reducingagent, and optionally a blocking group for the sulfhydryl groupsresulting from cleavage of disulfide linkages, to produce 3.5S Fab′monovalent fragments. Alternatively, an enzymatic cleavage using papainproduces two monovalent Fab fragments and an Fc fragment directly. Thesemethods are described, for example, by Goldenberg, U.S. Pat. Nos.4,036,945 and 4,331,647 and references contained therein, which patentsare incorporated herein in their entireties by reference. Also, seeNisonoff et al., Arch Biochem. Biophys. 89: 230 (1960); Porter, Biochem.J. 73: 119 (1959), Edelman et al., in METHODS IN ENZYMOLOGY VOL. 1, page422 (Academic Press 1967), and Coligan at pages 2.8.1-2.8.10 and2.10.-2.10.4.

[0062] Another form of an antibody fragment is a peptide coding for asingle complementarity-determining region (CDR). A CDR is a segment ofthe variable region of an antibody that is complementary in structure tothe epitope to which the antibody binds and is more variable than therest of the variable region. Accordingly, a CDR is sometimes referred toas hypervariable region. A variable region comprises three CDRs. CDRpeptides can be obtained by constructing genes encoding the CDR of anantibody of interest. Such genes are prepared, for example, by using thepolymerase chain reaction to synthesize the variable region from RNA ofantibody-producing cells. See, for example, Larrick et al., Methods: ACompanion to Methods in Enzymology 2: 106 (1991); Courtenay-Luck,“Genetic Manipulation of Monoclonal Antibodies,” in MONOCLONALANTIBODIES: PRODUCTION, ENGINEERING AND CLINICAL APPLICATION, Ritter etal. (eds.), pages 166-179 (Cambridge University Press 1995); and Ward etal., “Genetic Manipulation and Expression of Antibodies,” in MONOCLONALANTIBODIES: PRINCIPLES AND APPLICATIONS, Birch et al., (eds.), pages137-185 (Wiley-Liss, Inc. 1995).

[0063] Other methods of cleaving antibodies, such as separation of heavychains to form monovalent light-heavy chain fragments, further cleavageof fragments, or other enzymatic, chemical or genetic techniques mayalso be used, so long as the fragments bind to the antigen that isrecognized by the intact antibody.

[0064] Production of Chimeric, Humanized and Human RS7 Antibody FusionProteins

[0065] Antibody fusion proteins and fragments thereof can be prepared bya variety of conventional procedures, ranging from glutaraldehydelinkage to more specific linkages between functional groups. Theantibodies and/or antibody fragments are preferably covalently bound toone another, directly or through a linker moiety, through one or morefunctional groups on the antibody or fragment, e.g., amine, carboxyl,phenyl, thiol, or hydroxyl groups. Various conventional linkers inaddition to glutaraldehyde can be used, e.g., disiocyanates,diiosothiocyanates, bis(hydroxysuccinimide) esters, carbodiimides,maleimidehydroxysuccinimide esters, and the like.

[0066] A simple method to produce chimeric, humanized and human RS7antibody fusion proteins is to mix the antibodies or fragments in thepresence of glutaraldehyde to form an antibody fusion protein. Theinitial Schiff base linkages can be stabilized, e.g., by borohydridereduction to secondary amines. A diiosothiocyanates or carbodiimide canbe used in place of glutaraldehyde as a non-site-specific linker.Antibody fusion proteins are expected to have a greater bindingspecificity than MAbs, since the fusion proteins comprise moieties thatbind to at least two epitopes of the RS7 antigen. Thus, antibody fusionproteins are the preferred form of RS7 antigen binding protein fortherapy.

[0067] In the present context, an antibody fusion protein comprises atleast two chimeric, humanized or human RS7 MAbs, or fragments thereof,wherein at least two of the MAbs or fragments bind to different epitopesof the RS7 antigen or against an RS7 epitope and that of a totallydifferent antigen. For example, a bispecific RS7 antibody fusion proteinmay comprise a CEA antibody or fragment thereof and the RS7 MAb orfragment thereof. Such a bispecific RS7 antibody fusion protein can beprepared, for example, by obtaining an F(ab′)₂ fragment from CEA asdescribed above. The interchain disulfide bridges of the antibodyF(ab′)₂ fragment are gently reduced with cysteine, taking care to avoidlight-heavy chain linkage, to form Fab′-SH fragments. The SH group(s) is(are) activated with an excess of bis-maleimide linker(1,1′-(methylenedi-4,1-phenylene)bis-malemide). The RS7 MAb is convertedto Fab′-SH and then reacted with the activated CEA Fab′-SH fragment toobtain a bispecific RS7 antibody fusion protein.

[0068] A polyspecific RS7 antibody fusion protein can be obtained byadding RS7 antigen binding moieties to a bispecific chimeric, humanizedor human RS7 antibody fusion protein. For example, a bispecific antibodyfusion protein can be reacted with 2-iminothiolane to introduce one ormore sulfhydryl groups for use in coupling the bispecific fusion proteinto a third RS7 antigen MAb or fragment, using the bis-maleimideactivation procedure described above. These techniques for producingantibody composites are well known to those of skill in the art. See,for example, U.S. Pat. No. 4,925,648, which is incorporated by referencein its entirety.

[0069] Bispecific antibodies can be made by a variety of conventionalmethods, e.g., disulfide cleavage and reformation of mixtures of wholeIgG or, preferably F(ab′)₂ fragments, fusions of more than one hybridomato form polyomas that produce antibodies having more than onespecificity, and by genetic engineering. Bispecific antibody fusionproteins have been prepared by oxidative cleavage of Fab′ fragmentsresulting from reductive cleavage of different antibodies. This isadvantageously carried out by mixing two different F(ab′)₂ fragmentsproduced by pepsin digestion of two different antibodies, reductivecleavage to form a mixture of Fab′ fragments, followed by oxidativereformation of the disulfide linkages to produce a mixture of F(ab′)₂fragments including bispecific antibody fusion proteins containing aFab′ potion specific to each of the original epitopes. Generaltechniques for the preparation of antibody fusion proteins may be found,for example, in Nisonoff et al., Arch Biochem. Biophys. 93: 470 (1961),Hämmerling et al., J. Exp. Med. 128: 1461 (1968), and U.S. Pat. No.4,331,647. Contemplated in the present invention is an antibody fusionprotein or fragment thereof comprising at least one first anti-EGP-1 MAbor fragment thereof and at least one second MAb or fragment thereof,other than the anti-EGP-1 MAbs or fragments thereof of the presentinvention.

[0070] More selective linkage can be achieved by using aheterobifunctional linker such as maleimidehydroxysuccinimide ester.Reaction of the ester with an antibody or fragment will derivatize aminegroups on the antibody or fragment, and the derivative can then bereacted with, e.g., and antibody Fab fragment having free sulfhydrylgroups (or, a larger fragment or intact antibody with sulfhydryl groupsappended thereto by, e.g., Traut's Reagent). Such a linker is lesslikely to crosslink groups in the same antibody and improves theselectivity of the linkage.

[0071] It is advantageous to link the antibodies or fragments at sitesremote from the antigen binding sites. This can be accomplished by,e.g., linkage to cleaved interchain sulfydryl groups, as noted above.Another method involves reacting an antibody having an oxidizedcarbohydrate portion with another antibody, which has at lease one freeamine function. This results in an initial Schiff base (mime) linkage,which is preferably stabilized by reduction to a secondary amine, e.g.,by borohydride reduction, to form the final composite. Suchsite-specific linkages are disclosed, for small molecules, in U.S. Pat.No. 4,671,958, and for larger addends in U.S. Pat. No.4,699,784—incorporated by reference.

[0072] ScFvs with linkers greater than 12 amino acid residues in length(for example, 15-or 18-residue linkers) allow interacting between theV_(H) and V_(L) domains on the same chain and generally form a mixtureof monomers, dimers (termed diabodies) and small amounts of higher massmultimers, (Kortt et al., Eur. J. Biochem. (1994) 221: 151-157). ScFvswith linkers of 5 or less amino acid residues, however, prohibitintramolecular pairing of the V_(H) and V_(L) domains on the same chain,forcing pairing with V_(H) and V_(L) domains on a different chain.Linkers between 3- and 12-residues form predominantly dimers (Atwell etal., Protein Engineering (1999) 12: 597-604). With linkers between 0 and2 residues, trimeric (termed triabodies), tetrameric (termedtetrabodies) or higher oligomeric structures of scFvs are formed;however, the exact patterns of oligomerization appear to depend on thecomposition as well as the orientation of the V-domains, in addition tothe linker length. For example, scFvs of the anti-neuraminidase antibodyNC 10 formed predominantly trimers (V_(H) to V_(L) orientation) ortetramers (V_(L) to V_(H) orientation) with 0-residue linkers (Dolezalet al., Protein Engineering (2000) 13: 565-574). For scFvs constructedfrom NC10 with 1- and 2-residue linkers, the V_(H) to V_(L) orientationformed predominantly diabodies (Atwell et al., Protein Engineering(1999) 12: 597-604); in contrast, the V_(L) to V_(H) orientation formeda mixture of tetramers, trimers, dimers, and higher mass multimers(Dolezal et al., Protein Engineering (2000) 13: 565-574). For scFvsconstructed from the anti-CD19 antibody HD37 in the V_(H) to V_(L)orientation, the 0-residue linker formed exclusively trimers and the1-residue linker formed exclusively tetramers (Le Gall et al., FEBSLetters (1999) 453: 164-168).

[0073] The RS7 antibodies and fragments thereof of the present inventioncan also be used to produce antigen-specific diabodies, triabodies andtetrabodies, which are multivalent but monospecific. The non-covalentassociation of two or more scFv molecules can form functional diabodies,triabodies and tetrabodies. Monospecific diabodies are homodimers of thesame scFv, where each scFv comprises the V_(H) domain from the selectedantibody connected by a short linker to the V_(L) domain of the sameantibody. A diabody is a bivalent dimer formed by the non-covalentassociation of two scFvs, yielding two Fv binding sites. A triabodyresults from the formation of a trivalent trimer of three scFvs,yielding three binding sites, and a tetrabody is a tetravalent tetramerof four scFvs, resulting in four binding sites. Several monospecificdiabodies have been made using an expression vector that contains arecombinant gene construct comprising V_(H1)-linker-V_(L1). See Holligeret al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993); Atwell et al.,Molecular Immunology 33: 1301-1302 (1996); Holliger et al., NatureBiotechnology 15: 632-631(1997); Helfrich et al., Int. J. Cancer 76:232-239 (1998); Kipriyanov et al., Int. J Cancer 77: 763-772 (1998);Holiger et al., Cancer Research 59: 2909-2916(1999)). Methods ofconstructing scFvs are disclosed in U.S. Pat. No. 4,946,778 (1990) andU.S. Pat. No. 5,132,405 (1992). Methods of producing multivalent,monospecific binding proteins based on scFv are disclosed in U.S. Pat.No. 5,837,242 (1998), U.S. Pat. No. 5,844,094 (1998) and WO-98/44001(1998). A preferred embodiment of the instant invention is amultivalent, multispecific antibody or fragment thereof comprising oneor more antigen binding sites having affinity toward an EGP-1 targetantigen and one or more hapten binding sites having affinity towardshapten molecules.

[0074] Determining Antibody Binding Affinity

[0075] Comparative binding affinities of the mRS7, cRS7 and hRS7antibodies thus isolated may be determined by direct radioimmunoassay.RS7 can be labeled with ¹³¹I or ¹²⁵I using the chloramines-T method(see, for example, Greenwood et al., Biochem. J., 89: 123 (1963) whichis incorporated by reference). The specific activity of the iodinatedantibody is typically adjusted to about 10 μCi/μg. Unlabeled and labeledantibodies are diluted to the appropriate concentrations using reactionmedium (HSFM supplemented with 1% horse serum and 100 μg/ml gentamicin).The appropriate concentrations of both labeled and unlabeled antibodiesare added together to the reaction tubes in a total volume of 100 μl. Aculture of ME180 cells (a human cervical carcinoma cell line) is sampledand the cell concentration determined. The culture is centrifuged andthe collected cells washed once in reaction medium followed byresuspension in reaction medium to a final concentration of about 10⁷cells/ml. All procedures are carried out in the cold at 4° C. The cellsuspension, 100 μl, is added to the reaction tubes. The reaction iscarried out at 4° C. for 2 h with periodic gentle shaking of thereaction tubes to resuspend the cells. Following the reaction period, 5ml of wash buffer (PBS containing 1% BSA) is added to each tube. Thesuspension is centrifuged and the cell pellet washed a second time withanother 5 ml of wash buffer. Following centrifugation, the amount ofremaining radioactivity remaining in the cell pellet is determined in agamma counter (Minaxi, Packard Instruments, Sterling, Va.).

[0076] Expression Vectors

[0077] An expression vector is a DNA molecule comprising a gene that isexpressed in a host cell. Typically, gene expression is placed under thecontrol of certain regulatory elements, including constitutive orinducible promoters, tissue-specific regulatory elements, and enhancers.Such a gene is said to be “operably linked to” the regulatory elements.A promoter is a DNA sequence that directs the transcription of astructural gene. A structural gene is a DNA sequence that is transcribedinto messenger RNA (mRNA) which is then translated into a sequence ofamino acids characteristic of a specific polypeptide. Typically, apromoter is located in the 5′ region of a gene, proximal to thetranscriptional start site of a structural gene. If a promoter is aninducible promoter, then the rate of transcription increases in responseto an inducing agent. In contrast, the rate of transcription is notregulated by an inducing agent if the promoter is a constitutivepromoter. An enhancer is a DNA regulatory element that can increase theefficiency of transcription, regardless of the distance or orientationof the enhancer relative to the start site of transcription.

[0078] An isolated DNA molecule is a fragment of DNA that is notintegrated in the genomic DNA of an organism. For example, a cloned RS7antigen gene is a DNA fragment that has been separated from the genomicDNA of a mammalian cell. Another example of an isolated DNA molecule isa chemically-synthesized DNA molecule that is not integrated in thegenomic DNA of an organism. Complementary DNA (cDNA) is asingle-stranded DNA molecule that is formed from an mRNA template by theenzyme reverse transcriptase. Typically, a primer complementary toportions of mRNA is employed for the initiation of reversetranscription. Those skilled in the art also use the term “cDNA” torefer to a double-stranded DNA molecule consisting of such asingle-stranded DNA molecule and its complementary DNA strand.

[0079] A cloning vector is a DNA molecule, such as a plasmid, cosmid, orbacteriophage that has the capability of replicating autonomously in ahost cell. Cloning vectors typically contain one or a small number ofrestriction endonuclease recognition sites at which foreign DNAsequences can be inserted in a determinable fashion without loss of anessential biological function of the vector, as well as a marker genethat is suitable for use in the identification and selection of cellstransformed with the cloning vector. Marker genes typically includegenes that provide tetracycline resistance or ampicillin resistance. Arecombinant host may be any prokaryotic or eukaryotic cell that containseither a cloning vector or expression vector. This term also includesthose prokaryotic or eukaryotic cells that have been geneticallyengineered to contain the cloned gene(s) in the chromosome or genome ofthe host cell. The term expression refers to the biosynthesis of a geneproduct. For example, in the case of a structural gene, expressioninvolves transcription of the structural gene into mRNA and thetranslation of mRNA into one or more polypeptides.

[0080] Humanized, Human and Chimeric RS7 Antibodies use for Treatmentand Diagnosis

[0081] Contemplated in the present invention is a method of diagnosingor treating a malignancy in a subject comprising administering to thesubject a therapeutically effective amount of a therapeutic conjugatecomprising an EGP-1 MAb or fragment thereof or an antibody fusionprotein or fragment thereof, wherein the EGP-1MAb or fragment thereof orantibody fusion protein or fragment thereof is bound to at least onetherapeutic agent and then formulated in a pharmaceutically suitableexcipient. It is also contemplated that an unconjugated (naked) EGP-1MAb or fusion construct with other antigen-binding moieties also can besued as a therapeutic for cancer cells expressing EGP-1. Theseunconjugated antibodies may be given advantageously in combination withother therapeutic modalities, such as chemotherapy, radiotherapy, and/orimmunotherapy, either together or in various sequences and schedules.Also preferred is a method for diagnosing or treating cancer,comprising: administering a multivalent, multispecific antibody orfragment thereof comprising one or more antigen binding sites toward aEGP-1 antigen and one or more hapten binding sites to a subject in needthereof, waiting a sufficient amount of time for an amount of thenon-binding protein to clear the subject's blood stream; and thenadministering to the subject a carrier molecule comprising a diagnosticagent, a therapeutic agent, or a combination thereof, that binds to thebinding site of the multivalent, multispecific antibody or fragmentthereof. In a preferred embodiment, the cancer is a lung, breast, headand neck, ovarian, prostate, bladder or colon cancer.

[0082] Hybridoma technology for the production of monoclonal antibodies(MAbs) has provided a method for the production of molecular probescapable of locating or killing cancer cells. Tumor imaging techniquesusing radiolabeled MAbs have been used to delineate cancerous invasionin a number of malignancies. In experimental animals and in humans,antibodies have been used for the radioimmunodetection ofcarcinoembryonic antigen in diverse tumors that express carcinoembryonicantigen, and also tumors such as melanoma, colon carcinoma, and breastcarcinoma with other targeting antibodies. Goldenberg et al., CancerRes. 40: 2984 (1980); Hwang et al., Cancer Res. 45: 4150 (1985);Zalcberg et al., J. Nat'l Cancer Inst. 71: 801 (1983); Colcher et al.,Cancer Res. 43: 736 (1983); (Larson et al., J. Nucl. Med. 24: 123(1983); DeLand et al., Cancer Res. 40: 3046 (1980); Epenetos et al.,Lancet 2: 999 (1982).

[0083] The use of MAbs for in vitro diagnosis is well known. See, forexample, Carlsson et al., Bio/Technology 7 (6): 567 (1989). For example,MAbs can be used to detect the presence of a tumor-associated antigen intissue from biopsy samples. MAbs also can be used to measure the amountof tumor-associated antigen in clinical fluid samples using techniquessuch as radioimmunoassay, enzyme-linked immunosorbent assay, andfluorescence immunoassay.

[0084] Conjugates of tumor-targeted MAbs and toxins can be used toselectively kill cancer cells in vivo (Spalding, Bio/Technology 9(8):701 (1991); Goldenberg, Scientfic American Science & Medicine 1(1): 64(1994)). For example, therapeutic studies in experimental animal modelshave demonstrated the anti-tumor activity of antibodies carryingcytotoxic radionuclides. (Goldenberg et al., Cancer Res. 41: 4354(1981), Cheung et al., J. Nat'l Cancer Inst. 77: 739 (1986), andSenekowitsch et al., J. Nucl. Med. 30: 531 (1989)). Also, see Stein etal., Antibody Immunoconj. Radiopharm. 4: 703 (1991), which is fullyincorporated by reference. Moreover, Phase-I therapeutic trials withsome of these MAbs have been initiated for treatment of lymphoma,melanoma, and other malignancies. See, for example, DeNardo et al., Int.J. Cancer Suppl. 3: 96 (1988), and Goldenberg et al., J. Clin. Oncol. 9:548 (1991).

[0085] Humanized, chimeric and fully human antibodies and fragmentsthereof are suitable for use in therapeutic methods and diagnosticmethods. Accordingly, contemplated in the present invention is a methodof delivering a diagnostic or therapeutic agent, or a combinationthereof, to a target comprising (i) providing a composition thatcomprises an anti-EGP-1 antibody and (ii) administering to a subject inneed thereof the diagnostic or therapeutic antibody conjugate.Preferably, the chimeric, humanized and fully human RS7 antibodies andfragments thereof of the present invention are used in methods fortreating malignancies.

[0086] Also described herein is a cancer cell targeting diagnostic ortherapeutic conjugate comprising an antibody component comprising ananti-EGP-1 mAb or fragment thereof or an antibody fusion protein orfragment thereof that binds to the cancer cell, wherein the antibodycomponent is bound to at least one diagnostic or at least onetherapeutic agent. Preferably, the diagnostic conjugate comprises atleast a photoactive diagnostic agent or an MRI contrast agent. Stillpreferred, the diagnostic agent is a radioactive label with an energybetween 60 and 4,000 keV.

[0087] The compositions for treatment contain at least one naked orconjugated humanized, chimeric or human RS7 antibody alone, or incombination with other naked or conjugated humanized, chimeric, human orother antibodies of the present invention, or other naked or conjugatedhumanized, chimeric or human antibodies not disclosed herein. Thepresent invention also contemplates administration of a conjugated ornaked antibody with a therapeutic agent such as an immunomodulator, ordiagnostic agent that is not conjugated to the anti-EGP-1 antibody.Naked or conjugated antibodies to the same or different epitope orantigen may be also combined with one or more of the antibodies of thepresent invention.

[0088] Accordingly, the present invention contemplates theadministration anti-EGP-1 antibodies and fragments thereof alone, as anaked antibody or antibody fragment, or administered as a multimodaltherapy. Preferably, the antibody is a humanized, chimeric or fullyhuman RS7 antibody or fragment thereof. Multimodal therapies of thepresent invention further include immunotherapy with a naked anti-EGP-1antibody supplemented with administration of other antibodies in theform of naked antibodies, fusion proteins, or as immunoconjugates. Forexample, a humanized, chimeric or fully human RS7 antibody may becombined with another naked humanized, chimeric RS7 or other antibody,or a humanized, chimeric RS7 or other antibody conjugated to an isotope,one or more chemotherapeutic agents, cytokines, toxins or a combinationthereof. For example, the present invention contemplates treatment of anaked or conjugated EGP-1 or RS7 antibody or fragments thereof before,in combination with, or after other solid tumor/carcinoma associatedantibodies such as anti-EGP-2, CEA, CSAp, MUC1-4, EGFR, HER2/neu, PSA,CC49 (anti-Tag 72 antibody) and PSMA antibodies. These solid tumorantibodies may be naked or conjugated to, inter alia, drugs, enzymes,hormones, toxins, isotopes, or immunomodulators. A fusion protein of ahumanized, chimeric or fully human RS7 antibody and a toxin or may alsobe used in this invention. Many different antibody combinations may beconstructed, either as naked antibodies or as partly naked and partlyconjugated with a therapeutic agent or immunomodulator. Alternatively,different naked antibody combinations may be employed for administrationin combination with other therapeutic agents, such as a cytotoxic drugor with radiation. Combinations of such antibodies can also be made,advantageously, with antisense oligonucleotides, as are known in theart. As such, the therapeutic conjugates may comprise anoligonucleotide, especially an antisense oligonucleotide that preferablyare directed against oncogenes and oncogene products of B-cellmalignancies. For example, antisense molecules inhibiting bcl-2expression that are described in U.S. Pat. No. 5,734,033 (Reed) which isincorporated by reference in its entirety, may also be conjugated to, orform the therapeutic agent portion of an antibody fusion protein or beadministered with a humanized RS7 antibody of the present invention.

[0089] The monospecific binding proteins described herein that arelinked to diagnostic or therapeutic agents directly target RS7 positivetumors. The monospecific molecules bind selectively to targeted antigensand as the number of binding sites on the molecule increases, theaffinity for the target cell increases and a longer residence time isobserved at the desired location. Moreover, non-antigen bound moleculesare cleared from the body quickly and exposure of normal tissues isminimized. A use of multispecific binding proteins is pre-targeting RS7positive tumors for subsequent specific delivery of diagnostic ortherapeutic agents. The agents are carried by histamine succinyl glycyl(HSG) containing peptides. The murine monoclonal antibody designated 679(an IgG1, K) binds with high affinity to molecules containing thetri-peptide moiety, HSG (Morel et al, Molecular immunology, 27,995-1000, 1990). 679 MAb can form a bispecific binding protein with hRS7that binds with HSG and the target antigen. Alternative haptens may alsobe utilized. These binding proteins bind selectively to targetedantigens allowing for increased affinity and a longer residence time atthe desired location. Moreover, non-antigen bound diabodies are clearedfrom the body quickly and exposure of normal tissues is minimized.

[0090] RS7 antibodies and fragments thereof can be used to treatmammalian disorders such as cancer. The cancer includes, but is notlimited to, lung, breast, bladder, ovarian prostate and colon cancers.

[0091] Delivering a diagnostic or a therapeutic agent to a target fordiagnosis or treatment in accordance with the invention includesproviding the anti-EGP-1 antibody or fragments thereof with a diagnosticor therapeutic agent and administering to a subject in need thereof withthe binding protein. Diagnosis further requires the step of detectingthe bound proteins with known techniques.

[0092] Administration of the antibodies and their fragments of thepresent invention with diagnostic or therapeutic agents can be effectedin a mammal by intravenous, intraarterial, intraperitoneal,intramuscular, subcutaneous, intrapleural, intrathecal, perfusionthrough a regional catheter, or direct intralesional injection. Whenadministering the binding protein by injection, the administration maybe by continuous infusion or by single or multiple boluses. Doses in therange of 20 to 800 mg/m² are feasible, with doses between 100 and 500mg/m² preferably, for therapy, and commensurately lower dosesrecommended for diagnostic imaging, such as 0.5 mg to 100 mg/patient.Such doses may be repeated at different frequencies, depending on theclinical situation and patient tolerance.

[0093] The antibody with the diagnostic or therapeutic agent may beprovided as a kit for human or mammalian therapeutic and diagnostic usein a pharmaceutically acceptable injection vehicle, preferablyphosphate-buffered saline (PBS) at physiological pH and concentration.The preparation preferably will be sterile, especially if it is intendedfor use in humans. Optional components of such kits include stabilizers,buffers, labeling reagents, radioisotopes, paramagnetic compounds,second antibody for enhanced clearance, and conventional syringes,columns, vials and the like.

[0094] Naked Antibody Therapy

[0095] A therapeutically effective amount of the naked chimeric,humanized and fully human RS7 antibodies, or their fragments, can beformulated in a pharmaceutically acceptable excipient. The efficacy ofthe naked chimeric, humanized and fully human RS7antibodies can also beenhanced by supplementing these naked antibodies with one or more othernaked antibodies, with one or more immunoconjugates of chimeric,humanized and fully human RS7 antibodies conjugated to a therapeuticagent, such as a drug, toxin, immunomodulator, hormone, growth factor,enzyme or therapeutic radionuclides, or with one or more therapeuticagent, including a drug, toxin, immunomodulator, hormone, growth factor,enzyme, oligonucleotide, or therapeutic radionuclide, administeredconcurrently or sequentially or according to a prescribed dosingregimen, with the RS7 antibodies or fragments thereof.

[0096] In a preferred embodiment, the naked or conjugated RS7 antibodiesof the present invention are combined with at least one cancer drug.Such combination therapy can improve the effect of the drug or lowerdrug dose that is needed. For example, the IC₅₀ value was determined forDox-RS7 and 2P-Dox-RS7 on a lung cancer cell line, Calu3, and two breastcancer cell lines, MDA468 and T47D, respectively. Calu3 and T47D cellsare positive for an EGP-1 antigen and negative for a CEA antigen, andMDA468 is positive for both the EGP-1 and CEA antigens. Results indicatethat the IC₅₀ value for Dox-RS7 is 0.04 μg/ml and for 2P-Dox-RS7 is0.023 μg/ml. Therefore, conjugating a naked, human, humanized orchimeric anti-EGP-1 antibody or fragment of the present invention to aparticular drug, such as 2P-Dox may help overcome multidrug resistance.This is also possible when the antibody is given in a combination with aparticular drug, as described.

[0097] RS7 Immunoconjugates

[0098] The present invention also contemplates the use of humanized,chimeric and human RS7 antibodies and fragments thereof for therapy. Theobjective of immunotherapy is to deliver cytotoxic doses ofradioactivity, toxin, cytokine, enzyme, or hormone, or drug to targetcells, while minimizing exposure to non-target tissues. The RS7 antigenbinding proteins of the present invention can be used to treat a varietyof tumors, such as of the lung, breast, bladder, ovary, uterus, stomach,and prostate.

[0099] Any of the antibodies or antibody fusion proteins and fragmentsthereof of the present invention can be conjugated with one or moretherapeutic or diagnostic agents. Generally, one therapeutic ordiagnostic agent is attached to each antibody or antibody fragment butmore than one therapeutic agent or diagnostic agent can be attached tothe same antibody or antibody fragment. If the Fc region is absent (forexample when the antibody used as the antibody component of theimmunoconjugate is an antibody fragment), it is possible to introduce acarbohydrate moiety into the light chain variable region of afull-length antibody or antibody fragment. See, for example, Leung etal., J. Immunol. 154: 5919 (1995); Hansen et al., U.S. Pat. No.5,443,953 (1995), Leung et al., U.S. Pat. No. 6,254,868, all of whichare incorporated in their entirety by reference. The engineeredcarbohydrate moiety is used to attach the therapeutic or diagnosticagent.

[0100] Methods for conjugating peptides to antibody components via anantibody carbohydrate moiety are well-known to those of skill in theart. See, for example, Shih et al., Int. J. Cancer 41: 832 (1988); Shihet al., Int. J. Cancer 46: 1101 (1990); and Shih et al., U.S. Pat. No.5,057,313, all of which are incorporated in their entirety by reference.The general method involves reacting an antibody component having anoxidized carbohydrate portion with a carrier polymer that has at leastone free amine function and that is loaded with a plurality of peptide.This reaction results in an initial Schiff base (imine) linkage, whichcan be stabilized by reduction to a secondary amine to form the finalconjugate. Also, a chelator such as DTPA (such as Mx-DTPA), DOTA, TETA,or NOTA can be attached to the antibody.

[0101] The antibody fusion proteins of the present invention comprisetwo or more antibodies or fragments thereof and each of the antibodiesor fragments that compose this fusion protein can contain a therapeuticagent or diagnostic agent. Additionally, one or more of the antibodiesor fragments of the antibody fusion protein can have more than onetherapeutic of diagnostic agent attached. Further, the therapeuticagents do not need to be the same but can be different therapeuticagents, for example, one can attach a drug and a radioisotope to thesame fusion protein. Particularly, an IgG can be radiolabeled with ¹³¹Iand attached to a drug. The ¹³¹I can be incorporated into the tyrosineof the IgG and the drug attached to the epsilon amino group of the IgGlysines. Both therapeutic and diagnostic agents also can be attached toreduced SH groups and to the carbohydrate side chains.

[0102] A wide variety of diagnostic and therapeutic reagents can beadvantageously conjugated to the antibodies of the invention. Thetherapeutic agents recited here are those agents that also are usefulfor administration separately with the naked antibody as describedabove. Therapeutic agents include, for example, chemotherapeutic drugssuch as vinca alkaloids, anthracyclines, epidophyllotoxinw, taxanes,antimetabolites, alkylating agents, antibiotics, substituted urea,enzymes, Cox-2 inhibitors, antimitotics, antiangiogenic and apoptotoicagents, particularly doxorubicin, doxorubicin analogs, methotrexate,taxol, CPT-11, camptothecans, and others from these and other classes ofanticancer agents, methyl hydrazine derivative, adrenocorticalsuppressant, antagonist, endostatin, taxol, and the like. Other usefulcancer chemotherapeutic drugs for the preparation of immunoconjugatesand antibody fusion proteins include nitrogen mustards, ethyleniminederivatives, alkyl sulfonates, nitrosoureas, triazenes, folic acidanalogs, COX-2 inhibitors, pyrimidine analogs, purine analogs, platinumcoordination complexes, hormones, tyrosine kinase inhibitors, such asthose that inhibit a EGF-receptor tyrosine kinase, a BCR ABL tyrosinekinase or a VEGF-receptor tyrosine kinase, and the like. Suitablechemotherapeutic agents are described in REMINGTON'S PHARMACEUTICALSCIENCES, 19th Ed. (Mack Publishing Co. 1995), and in GOODMAN ANDGILMAN'S THE PHARMACOLOGICAL BASIS OF THERAPEUTICS, 7th Ed. (MacMillanPublishing Co. 1985), as well as revised editions of these publications.Other suitable chemotherapeutic agents, such as experimental drugs, areknown to those of skill in the art.

[0103] A toxin, such as Pseudomonas exotoxin, may also be complexed toor form the therapeutic agent portion of an immunoconjugate of the RS7and hRS7 antibodies of the present invention. Other toxins suitablyemployed in the preparation of such conjugates or other fusion proteins,include ricin, abrin, ribonuclease (RNase), DNase I, Staphylococcalenterotoxin-A, pokeweed antiviral protein, gelonin, diphtherin toxin,Pseudomonas exotoxin, and Pseudomonas endotoxin. See, for example,Pastan et al., Cell 47:641 (1986), and Goldenberg, Calif.—A CancerJournal for Clinicians 44:43 (1994). Additional toxins suitable for usein the present invention are known to those of skill in the art and aredisclosed in U.S. Pat. No. 6,077,499, which is incorporated in itsentirety by reference.

[0104] An immunomodulator, such as a cytokine may also be conjugated to,or form the therapeutic agent portion of the EGP-1, RS7 and hRS7immunoconjugate, or be administered unconjugated to the chimeric,humanized or human RS7 antibodies or fragments thereof of the presentinvention. As used herein, the term “immunomodulator” includescytokines, stem cell growth factors, lymphotoxins, such as tumornecrosis factor (TNF), and hematopoietic factors, such as interleukins(e.g., interleukin-1 (IL-1), IL-2, IL-3, IL-6, IL-10, IL-12, IL-18, andIL-21), colony stimulating factors (e.g., granulocyte-colony stimulatingfactor (G-CSF) and granulocyte macrophage-colony stimulating factor(GM-CSF)), interferons (e.g., interferons-α, -β, and -γ), the stem cellgrowth factor designated “S1 factor,” erythropoietin and thrombopoietin,or a combination thereof. Examples of suitable immunomodulator moietiesinclude IL-2, IL-6, IL-10, IL-12, IL-18, IL-21, interferon-γ, TNF-α, andthe like. Alternatively, subjects can receive naked EGP-1 or RS7antibodies and a separately administered cytokine, which can beadministered before, concurrently or after administration of the nakedRS7 antibodies. The RS7 antibody may also be conjugated to theimmunomodulator. The immunomodulator may also be conjugated to a hybridantibody consisting of one or more antibodies binding to differentantigens.

[0105] A therapeutic or diagnostic agent can be attached at the hingeregion of a reduced antibody component via disulfide bond formation. Asan alternative, such peptides can be attached to the antibody componentusing a heterobifunctional cross-linker, such as N-succinyl3-(2-pyridyldithio)proprionate (SPDP). Yu et al., Int. J. Cancer 56: 244(1994). General techniques for such conjugation are well known in theart. See, for example, Wong, CHEMISTRY OF PROTEIN CONJUGATION ANDCROSS-LINKING (CRC Press 1991); Upeslacis et al., “Modification ofAntibodies by Chemical Methods,” in MONOCLONAL ANTIBODIES: PRINCIPLESAND APPLICATIONS, Birch et al. (eds.), pages 187-230 (Wiley-Liss, Inc.1995); Price, “Production and Characterization of SyntheticPeptide-Derived Antibodies,” in MONOCLONAL ANTIBODIES: PRODUCTION,ENGINEERING AND CLINICAL APPLICATION, Ritter et al. (eds.), pages 60-84(Cambridge University Press 1995). Alternatively, the therapeutic ordiagnostic agent can be conjugated via a carbohydrate moiety in the Feregion of the antibody. The carbohydrate group can be used to increasethe loading of the same peptide that is bound to a thiol group, or thecarbohydrate moiety can be used to bind a different peptide.

[0106] Furthermore, a radiolabeled antibody, immunoconjugate, orfragments thereof may comprise a γ-emitting radioisotope or apositron-emitter useful for diagnostic imaging. Suitable radioisotopes,particularly in the energy range of 25 to 4,000 keV, include ¹³¹I, ¹²³I,¹²⁴I, ⁸⁶Y, ⁶²Cu, ⁶⁴Cu, ⁶⁷Ga, ⁶⁸Ga, ^(99m)Tc, ^(94m)Tc, ¹⁸F, ¹¹C, ¹³N,¹⁵O, ⁷⁵Br, and the like. See for example, U.S. Patent Applicationentitled “Labeling Targeting Agents with Gallium-68”—Inventors G.L.Griffiths and W. J. McBride, (U.S. Provisional Application No.60/342,104), which discloses positron emitters, such as ¹⁸F, ⁶⁸Ga,^(94m)Tc and the like, for imaging purposes and which is incorporated inits entirety by reference. Preferably, the energy range for diagnosticand therapeutic radionuclides is 25-4,000_keV. Other usefulradionuclides include ⁹⁰Y, ¹¹¹In, ¹²⁵I, ³H, ³⁵S, ¹⁴C, ¹⁸⁶Re, ¹⁸⁸Re,¹⁸⁹Re, ¹⁷⁷Lu, ⁶⁷Cu, ²¹²Bi, ²¹³Bi, ²¹¹At, ¹⁹⁸Au, ²²⁴Ac, ¹²⁶I, ¹³³I, ⁷⁷Br,^(113m)In, ⁹⁵Ru, ⁹⁷Ru, ¹⁰³Ru, ¹⁰⁵Ru, ¹⁰⁷Hg, ²⁰³Hg, ^(94m)Tc, ^(121m)Te,^(121m)Te, ^(125m)Te, ¹⁶⁵Tm, ¹⁶⁷Tm, ¹⁶⁸Tm, ¹¹¹Ag, ¹⁹⁷Pt, ¹⁰⁹Pd, ³²P,³³P, ⁴⁷Sc, ¹⁵³Sm, ¹⁷⁷Lu, ¹⁰⁵Rh, ¹⁴²Pr, ¹⁴³Pr, ¹⁶¹Tb, ¹⁶⁶Ho, ¹⁹⁹Au, ⁵⁷Co,⁵⁸Co, ⁵¹Cr, ⁵⁹Fe, ¹⁸F, ⁷⁵Se, ²⁰¹Tl, ²²⁵Ac, ⁷⁶Br, ⁸⁶Y, ¹⁶⁹Yb, ¹⁶⁶Dy,²¹²Pb, and ²²³Ra.

[0107] For example, ⁶⁷Cu, considered one of the more promisingradioisotopes for radioimmunotherapy due to its 61.5 hour half-life andabundant supply of beta particles and gamma rays, can be conjugated toan RS7 antigen binding protein using the chelating agent,p-bromoacetamido-benzyl-tetraethylaminetetraacetic acid (TETA). Chase,supra. Alternatively, ⁹⁰Y, which emits an energetic beta particle, canbe coupled to an RS7 antigen binding protein usingdiethylenetriaminepentaacetic acid (DTPA). Moreover, a method for thedirect radiolabeling of the RS7 MAb with ¹³¹I is described by Stein etal. (1991), supra, and the patent by Govindan et al., WO 9911294A1entitled “Stable Radioiodine Conjugates and Methods for TheirSynthesis,” and is incorporated herein by reference in their entirety.

[0108] The RS7 antibodies or fragments thereof of the present inventionthat have a boron addend-loaded carrier for thermal neutron activationtherapy will normally be effected in similar ways. However, it will beadvantageous to wait until non-targeted RS7 immunoconjugate clearsbefore neutron irradiation is performed. Clearance can be acceleratedusing an antibody that binds to the RS7 antibody. See U.S. Pat. No.4,624,846 for a description of this general principle. For example,boron addends such as carboranes, can be attached to RS7 antibodies.Carboranes can be prepared with carboxyl functions on pendant sidechains, as is well known in the art. Attachment of carboranes to acarrier, such as aminodextran, can be achieved by activation of thecarboxyl groups of the carboranes and condensation with amines on thecarrier. The intermediate conjugate is then conjugated to the RS7antibody. After administration of the RS7 antibody conjugate, a boronaddend is activated by thermal neutron irradiation and converted toradioactive atoms that decay by α-emission to produce highly toxic,short-range effects.

[0109] Furthermore, the present invention includes methods of diagnosingcancer in a subject. Diagnosis may be accomplished by administering adiagnostically effective amount of a diagnostic conjugate, formulated ina pharmaceutically suitable excipient, and detecting said label. Forexample, radioactive and non-radioactive agents can be used asdiagnostic agents. A suitable non-radioactive diagnostic agent is acontrast agent suitable for magnetic resonance imaging, computedtomography or ultrasound. Magnetic imaging agents include, for example,non-radioactive metals, such as manganese, iron and gadolinium,complexed with metal-chelate combinations that include 2-benzyl-DTPA andits monomethyl and cyclohexyl analogs, when used along with theantibodies of the invention. See U.S. Ser. No. 09/921,290 filed on Oct.10, 2001, which is incorporated in its entirety by reference.

[0110] Accordingly, a method of diagnosing a malignancy in a subject isdescribed, comprising (i) performing an in vitro diagnosis assay on aspecimen from the subject with a composition comprising a nakedanti-EGP-1 MAb or fragment thereof or a naked antibody fusion protein orfragment thereof. For example, RT-PCR and immunoassay in vitro diagnosismethods can be used to detect the presence of minute amounts of EGP-1 intissues, blood and other body fluids as a useful diagnostic/detectionmethod. Immunohistochemistry can be used to detect the presence of EGP-1in a cell or tissue. Preferably, the malignancy that is being diagnosedis a cancer. Most preferably, the cancer is selected from the group oflung, prostate, ovarian, breast, colon and bladder.

[0111] Additionally, a chelator such as DTPA, DOTA, TETA, or NOTA or asuitable peptide, to which a detectable label, such as a fluorescentmolecule, or cytotoxic agent, such as a heavy metal or radionuclide, canbe conjugated. For example, a therapeutically useful immunoconjugate canbe obtained by conjugating a photoactive agent or dye to an antibodyfusion protein. Fluorescent compositions, such as fluorochrome, andother chromogens, or dyes, such as porphyrins sensitive to visiblelight, have been used to detect and to treat lesions by directing thesuitable light to the lesion. In therapy, this has been termedphotoradiation, phototherapy, or photodynamic therapy (Jori et al.(eds.), PHOTODYNAMIC THERAPY OF TUMORS AND OTHER DISEASES (LibreriaProgetto 1985); van den Bergh, Chem. Britain 22:430 (1986)). Moreover,monoclonal antibodies have been coupled with photoactivated dyes forachieving phototherapy. Mew et al., J. Immunol. 130:1473 (1983); idem.,Cancer Res. 45:4380 (1985); Oseroff et al., Proc. Natl. Acad. Sci. USA83:8744 (1986); idem., Photochem. Photobiol. 46:83 (1987); Hasan et al.,Prog. Clin. Biol. Res. 288:471 (1989); Tatsuta et al., Lasers Surg. Med.9:422 (1989); Pelegrin et al., Cancer 67:2529 (1991). However, theseearlier studies did not include use of endoscopic therapy applications,especially with the use of antibody fragments or subfragments. Thus, thepresent invention contemplates the therapeutic use of immunoconjugatescomprising photoactive agents or dyes.

[0112] Contrast agents such as a MRI contrast agent, a paragmagnetic ionand an ultrasound enhancing agent are also contemplated in the presentinvention. For example, gadolinium ions, lanthanum ions, manganese ionsor other comparable label, CT contrast agents, and ultrasound contrastagents are suitable for use in the present invention. In a preferredembodiment, the ultrasound enhancing agent is a liposome that comprisesa humanized RS7 IgG or fragment thereof. Also preferred, the liposome isgas filled.

[0113] For purposes of therapy, the RS7 antibodies and fragments thereofof the present invention are administered to a patient in atherapeutically effective amount. An antibody is said to be administeredin a “therapeutically effective amount” if the amount administered isphysiologically significant. An agent is physiologically significant ifits presence results in a detectable change in the physiology of arecipient patient.

[0114] In vitro Diagnosis

[0115] The present invention contemplates the use of RS7 antibodies,including RS7 and hRS7 antibodies and fragments thereof, to screenbiological samples in vitro for the presence of the RS7 antigen. In suchimmunoassays, the RS7 antibody may be utilized in liquid phase or boundto a solid-phase carrier, as described below. Also, see Stein et al.(1993), supra, and Stein et al., Cancer Res. 49: 32 (1989), which isfully incorporated by reference.

[0116] One example of a screening method for determining whether abiological sample contains the RS7 antigen is the radioimmunoassay(RIA). For example, in one form of RIA, the substance under test ismixed with RS7 antigen MAb in the presence of radiolabeled RS7 antigen.In this method, the concentration of the test substance will beinversely proportional to the amount of labeled RS7 antigen bound to theMAb and directly related to the amount of free labeled RS7 antigen.Other suitable screening methods will be readily apparent to those ofskill in the art.

[0117] Alternatively, in vitro assays can be performed in which an RS7antigen binding protein is bound to a solid-phase carrier. For example,MAbs can be attached to a polymer, such as aminodextran, in order tolink the MAb to an insoluble support such as a polymer-coated bead, aplate or a tube.

[0118] Other suitable in vitro assays will be readily apparent to thoseof skill in the art. The specific concentrations of detectably labeledRS7 antigen binding protein and RS7 antigen, the temperature and time ofincubation, as well as other assay conditions may be varied, dependingon various factors including the concentration of the RS7 antigen in thesample, the nature of the sample, and the like. The binding activity ofa sample of RS7 antigen binding protein may be determined according towell known methods. Those skilled in the art will be able to determineoperative and optimal assay conditions for each determination byemploying routine experimentation.

[0119] Other such steps as washing, stirring, shaking, filtering and thelike may be added to the assays as is customary or necessary for theparticular situation.

[0120] The presence of the RS7 antigen in a biological sample can bedetermined using an enzyme-linked immunosorbent assay (ELISA). In thedirect competitive ELISA, a pure or semipure antigen preparation isbound to a solid support that is insoluble in the fluid or cellularextract being tested and a quantity of detectably labeled solubleantibody is added to permit detection and/or quantitation of the binarycomplex formed between solid-phase antigen and labeled antibody.

[0121] In contrast, a “double-determinant” ELISA, also known as a“two-site ELISA” or “sandwich assay,” requires small amounts of antigenand the assay does not require extensive purification of the antigen.Thus, the double-determinant ELISA is preferred to the directcompetitive ELISA for the detection of an antigen in a clinical sample.See, for example, the use of the double-determinant ELISA forquantitation of the c-myc oncoprotein in biopsy specimens. Field et al.,Oncogene 4: 1463 (1989); Spandidos et al., AntiCancer Res. 9: 821(1989).

[0122] In a double-determinant ELISA, a quantity of unlabeled MAb orantibody fragment (the “capture antibody”) is bound to a solid support,the test sample is brought into contact with the capture antibody, and aquantity of detectably labeled soluble antibody (or antibody fragment)is added to permit detection and/or quantitation of the ternary complexformed between the capture antibody, antigen, and labeled antibody. Anantibody fragment is a portion of an antibody such as F(ab′)₂, F(ab)₂,Fab′, Fab, and the like. In the present context, an antibody fragment isa portion of an RS7 MAb that binds to an epitope of the RS7 antigen. Theterm “antibody fragment” also includes any synthetic or geneticallyengineered protein that acts like an antibody by binding to a specificantigen to form a complex. For example, antibody fragments includeisolated fragments consisting of the light chain variable region, “Fv”fragments consisting of the variable regions of the heavy and lightchains, and recombinant single chain polypeptide molecules in whichlight and heavy variable regions are connected by a peptide linker. Anantibody fusion protein is a polyspecific antibody compositioncomprising at least two substantially monospecific antibodies orantibody fragments, wherein at least two of the antibodies or antibodyfragments bind to different epitopes of the RS7 antigen. An RS7 fusionprotein also includes a conjugate of an antibody fusion protein with adiagnostic or therapeutic agent. The term RS7 antibody includeshumanized, chimeric, human and murine antibodies, antibody fragmentsthereof, immunoconjugates and fragments thereof and antibody fusionproteins and fragments thereof.

[0123] Methods of performing a double-determinant ELISA are well-known.See, for example, Field et al., supra, Spandidos et al., supra, andMoore et al., “Twin-Site ELISAs for fos and myc Oncoproteins Using theAMPAK System,” in METHODS IN MOLECULAR BIOLOGY, VOL. 10, pages 273-281(The Humana Press, Inc. 1992). For example, in one method for thedetection of RS7 antigen using the double-determinant ELISA, finelyminced tissue from a biopsy sample is lyophilized and resuspended inlysis buffer (100 mM NaCl, 50 mM Tris-HCl, pH 7.4) containing 1%nonidet-p40 (NP40), 0.6 μl/ml aprotinin, 0.2 mM phenyl methyl sulphonylfluoride, 0.1 μg/ml leupeptin and 1 mM EDTA at a concentration of 10-20mg tissue (wet weight) per 500 μl solution. The suspension is incubatedfor 60 minutes on ice, and then sonicated for approximately six10-second intervals. Insoluble material is removed by centrifugation.

[0124] The soluble extract is added to microtiter plate wells containingan adsorbed RS7 antigen MAb as the capture antibody. Captured RS7antigen is then recognized by a second RS7 antigen MAb, which has beencoupled with alkaline phosphatase. The amount of bound alkalinephosphatase, proportional to the amount of RS7 antigen in the extract,is detected colormetrically using a chromogenic substrate, such asp-nitrophenylphosphate.

[0125] Alternatively, a double-determinant ELISA for the RS7 antigen canbe performed using horse radish peroxidase. Other variations of samplepreparation and the double-determinant ELISA can be devised by those ofskill in the art with routine experimentation.

[0126] In the double-determinant ELISA, the soluble antibody or antibodyfragment must bind to an RS7 epitope that is distinct from the epitoperecognized by the capture antibody. For example, the soluble antibodycan be the RS7 MAb, while the capture antibody can be MR23.Alternatively, the soluble antibody can be MR23, while the captureantibody can be the RS7 MAb.

[0127] The double-determinant ELISA can be performed to ascertainwhether the RS7 antigen is present in a biopsy sample. Alternatively,the assay can be performed to quantitate the amount of RS7 antigen thatis present in a clinical sample of body fluid. The quantitative assaycan be performed by including dilutions of purified RS7 antigen. Amethod for purifying the RS7 antigen is illustrated below.

[0128] The RS7 MAbs and fragments thereof of the present invention alsoare suited for the preparation of an assay kit. Such a kit may comprisea carrier means that is compartmentalized to receive in closeconfinement one or more container means such as vials, tubes and thelike, each of said container means comprising the separate elements ofthe immunoassay.

[0129] For example, there may be a container means containing thecapture antibody immobilized on a solid phase support, and a furthercontainer means containing detectably labeled antibodies in solution.Further container means may contain standard solutions comprising serialdilutions of RS7 antigen. The standard solutions of RS7 antigen may beused to prepare a standard curve with the concentration of RS7 antigenplotted on the abscissa and the detection signal on the ordinate. Theresults obtained from a sample containing RS7 antigen may beinterpolated from such a plot to give the concentration of RS7 antigenin the biological sample.

[0130] RS7 antibodies and their fragments of the present invention alsocan be used to detect the presence of the RS7 antigen in tissue sectionsprepared from a histological specimen. Such in situ detection can beused to determine the presence of the RS7 antigen and to determine thedistribution of the RS7 antigen in the examined tissue. In situdetection can be accomplished by applying a detectably-labeled RS7antigen binding protein to frozen tissue sections. Studies indicate thatthe RS7 antigen is not preserved in paraffin-embedded sections. Stein etal. (1993), supra. General techniques of in situ detection are wellknown to those of ordinary skill. See, for example, Ponder, “CellMarking Techniques and Their Application,” in MAMMALIAN DEVELOPMENT: APRACTICAL APPROACH 113-38 Monk (ed.) (IRL Press 1987), and Coligan atpages 5.8.1-5.8.8. Also, see Stein et al. (1989), supra, and Stein etal. (1993), supra.

[0131] RS7 antibodies and their fragments can be detectably labeled withany appropriate detection agent, for example, a radioisotope, an enzyme,a fluorescent label, a chemiluminescent label, a bioluminescent label ora paramagnetic label. Methods of making and detecting suchdetectably-labeled RS7 antigen binding proteins are well-known to thoseof ordinary skill in the art, and are described in more detail below.

[0132] The marker moiety can be a radioisotope that is detected by suchmeans as the use of a gamma counter or a scintillation counter or byautoradiography. In a preferred embodiment, the diagnostic conjugate isa gamma-, beta- or a positron-emitting isotope. A marker moiety in thepresent description refers to molecule that will generate a signal underpredetermined conditions. Examples of marker moieties includeradioisotopes, enzymes, fluorescent labels, chemiluminescent labels,bioluminescent labels and paramagnetic labels. As used herein, adiagnostic or therapeutic agent is a molecule or atom, which isconjugated to an antibody moiety to produce a conjugate, which is usefulfor diagnosis and for therapy. Examples of diagnostic or therapeuticagents include drugs, toxins, chelators, dyes, chromagens, boroncompounds, and marker moieties. Isotopes that are particularly usefulfor the purpose of the present invention are ³H, ¹³¹I, ³⁵S, ¹⁴C, andpreferably ¹²⁵I. Examples of other radionuclides are, for example, ⁹⁰Y,¹¹¹In, ^(99m)Tc, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁷⁷Lu, ⁶⁷Cu, ²¹²Bi, ²¹³Bi, and ²¹¹At.Additional radionuclides are also available as diagnostic andtherapeutic agents. Suitable diagnostic imaging isotopes are usually inthe range of 25 to 4,000 keV, while suitable therapeutic radionuclidesare usually in the range of 60 to 700 keV.

[0133] The RS7 antibodies and their fragments of the present inventionalso can be labeled with a fluorescent compound. The presence of afluorescently-labeled MAb is determined by exposing the RS7 antigenbinding protein to light of the proper wavelength and detecting theresultant fluorescence. Fluorescent labeling compounds includefluorescein isothiocyanate, rhodamine, phycoerytherin, phycocyanin,allophycocyanin, o-phthaldehyde and fluorescamine. Fluorescently-labeledRS7 antigen binding proteins are particularly useful for flow cytometryanalysis.

[0134] Alternatively, RS7 antibodies and their fragments can bedetectably labeled by coupling the RS7 antigen binding protein to achemiluminescent compound. The presence of the chemiluminescent-taggedMAb is determined by detecting the presence of luminescence that arisesduring the course of a chemical reaction. Examples of chemiluminescentlabeling compounds include luminol, isoluminol, an aromatic acridiniumester, an imidazole, an acridinium salt and an oxalate ester.

[0135] Similarly, a bioluminescent compound can be used to label RS7antibodies and fragments thereof the present invention. Bioluminescenceis a type of chemiluminescence found in biological systems in which acatalytic protein increases the efficiency of the chemiluminescentreaction. The presence of a bioluminescent protein is determined bydetecting the presence of luminescence. Bioluminescent compounds thatare useful for labeling include luciferin, luciferase and aequorin.

[0136] Alternatively, RS7 antibodies and fragments thereof can bedetectably labeled by linking the RS7 antibody to an enzyme. When theRS7 antibody-enzyme conjugate is incubated in the presence of theappropriate substrate, the enzyme moiety reacts with the substrate toproduce a chemical moiety, which can be detected, for example, byspectrophotometric, fluorometric or visual means. Examples of enzymesthat can be used to detectably label RS7 antibody include malatedehydrogenase, staphylococcal nuclease, delta-V-steroid isomerase, yeastalcohol dehydrogenase, α-glycerophosphate dehydrogenase, triosephosphate isomerase, horseradish peroxidase, alkaline phosphatase,asparaginase, glucose oxidase, β-galactosidase, ribonuclease, urease,catalase, glucose-6-phosphate dehydrogenase, glucoamylase andacetylcholinesterase.

[0137] RS7 antibodies, fusion proteins, and fragments thereof also canbe labeled with paramagnetic ions for purposes of in vivo diagnosis.Contrast agents that are particularly useful for magnetic resonanceimaging comprise Gd, Mn, Dy or Fe ions. RS7 antibodies and fragmentsthereof can also be conjugated to ultrasound contrast/enhancing agents.For example, the ultrasound contrast agent is a liposome that comprisesa humanized RS7 IgG or fragment thereof. Also preferred, the ultrasoundcontrast agent is a liposome that is gas filled.

[0138] In a related vein, a bispecific antibody can be conjugated to acontrast agent. For example, the bispecific antibody may comprise morethan one image-enhancing agent for use in ultrasound imaging. In apreferred embodiment, the contrast agent is a liposome. Preferably, theliposome comprises a bivalent DTPA-peptide covalently attached to theoutside surface of the liposome. Still preferred, the liposome is gasfilled.

[0139] Those of skill in the art will know of other suitable labels thatcan be employed in accordance with the present invention. The binding ofmarker moieties to RS7 antibodies can be accomplished using standardtechniques known to the art. Typical methodology in this regard isdescribed by Kennedy etal, Clin. Chim. Acta 70: 1 (1976), Schurs etal.,Clin. Chim. Acta 81: 1 (1977), Shih etal., Int'l J. Cancer 46: 1101(1990), Stein et al. (1990), supra, and Stein et al. (1993), supra.Also, see generally, Coligan.

[0140] The above-described in vitro and in situ detection methods may beused to assist in the diagnosis or staging of a pathological condition.For example, such methods can be used to detect tumors that express theRS7 antigen including tumors of the lung, breast, bladder, ovary,uterus, stomach, and prostate.

[0141] In vivo Diagnosis

[0142] The present invention also contemplates the use of RS7 antibodiesfor in vivo diagnosis. The method of diagnostic imaging withradiolabeled MAbs is well-known. In the technique of immunoscintigraphy,for example, antibodies are labeled with a gamma-emitting radioisotopeand introduced into a patient. A gamma camera is used to detect thelocation and distribution of gamma-emitting radioisotopes. See, forexample, Srivastava (ed.), RADIOLABELED MONOCLONAL ANTIBODIES FORIMAGING AND THERAPY (Plenum Press 1988), Chase, “Medical Applications ofRadioisotopes,” in REMINGTON'S PHARMACEUTICAL SCIENCES, 18th Edition,Gennaro et al. (eds.), pp. 624-652 (Mack Publishing Co., 1990), andBrown, “Clinical Use of Monoclonal Antibodies,” in BIOTECHNOLOGY ANDPHARMACY 227-49, Pezzuto et al. (eds.) (Chapman & Hall 1993).

[0143] For diagnostic imaging, radioisotopes may be bound to the RS7antibody either directly, or indirectly by using an intermediaryfunctional group. Useful intermediary functional groups includechelators such as ethylenediaminetetraacetic acid anddiethylenetriaminepentaacetic acid. For example, see Shih et al., supra,and U.S. Pat. No. 5,057,313.

[0144] The radiation dose delivered to the patient is maintained at aslow a level as possible through the choice of isotope for the bestcombination of minimum half-life, minimum retention in the body, andminimum quantity of isotope, which will permit detection and accuratemeasurement. Examples of radioisotopes that can be bound to RS7 antibodyand are appropriate for diagnostic imaging include ^(99m)Tc and ¹¹¹In.

[0145] Pharmaceutically Suitable Excipient

[0146] Additional pharmaceutical methods may be employed to control theduration of action of an RS7 antibody in a therapeutic application.Control release preparations can be prepared through the use of polymersto complex or adsorb the RS7 antibody. For example, biocompatiblepolymers include matrices of poly(ethylene-co-vinyl acetate) andmatrices of a polyanhydride copolymer of a stearic acid dimer andsebacic acid. Sherwood et al., Bio/Technology 10: 1446 (1992). The rateof release of an RS7 antibody from such a matrix depends upon themolecular weight of the RS7 antibody, the amount of RS7 antibody withinthe matrix, and the size of dispersed particles. Saltzman et al.,Biophys. J. 55: 163 (1989); Sherwood et al., supra. Other solid dosageforms are described in REMINGTON'S PHARMACEUTICAL SCIENCES, 18th ed.(1990).

[0147] The humanized, chimeric and human RS7 antibodies to be deliveredto a subject can consist of the antibody alone, immunoconjugate, fusionprotein, or can comprise one or more pharmaceutically suitableexcipients, one or more additional ingredients, or some combination ofthese.

[0148] The immunoconjugate, naked antibody, fusion protein, andfragments thereof of the present invention can be formulated accordingto known methods to prepare pharmaceutically useful compositions,whereby the immunoconjugate or naked antibody is combined in a mixturewith a pharmaceutically suitable excipient. Sterile phosphate-bufferedsaline is one example of a pharmaceutically suitable excipient. Othersuitable excipients are well known to those in the art. See, forexample, Ansel et al., PHARMACEUTICAL DOSAGE FORMS AND DRUG DELIVERYSYSTEMS, 5th Edition (Lea & Febiger 1990), and Gennaro (ed.),REMINGTON'S PHARMACEUTICAL SCIENCES, 18th Edition (Mack PublishingCompany 1990), and revised editions thereof.

[0149] The immunoconjugate or naked antibody of the present inventioncan be formulated for intravenous administration via, for example, bolusinjection or continuous infusion. Formulations for injection can bepresented in unit dosage form, e.g., in ampules or in multi-dosecontainers, with an added preservative. The compositions can take suchforms as suspensions, solutions or emulsions in oily or aqueousvehicles, and can contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredient can be in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use.

[0150] Additional pharmaceutical methods may be employed to control theduration of action of the therapeutic or diagnostic conjugate or nakedantibody. Control release preparations can be prepared through the useof polymers to complex or adsorb the immunoconjugate or naked antibody.For example, biocompatible polymers include matrices ofpoly(ethylene-co-vinyl acetate) and matrices of a polyanhydridecopolymer of a stearic acid dimer and sebacic acid. Sherwood et al.,Bio/Technology 10: 1446 (1992). The rate of release of animmunoconjugate or antibody from such a matrix depends upon themolecular weight of the immunoconjugate or antibody, the amount ofimmunoconjugate, antibody within the matrix, and the size of dispersedparticles. Saltzman et al., Biophys. J. 55: 163 (1989); Sherwood et al.,supra. Other solid dosage forms are described in Ansel et al.,PHARMACEUTICAL DOSAGE FORMS AND DRUG DELIVERY SYSTEMS, 5th Edition (Lea& Febiger 1990), and Gennaro (ed.), REMINGTON'S PHARMACEUTICAL SCIENCES,18th Edition (Mack Publishing Company 1990), and revised editionsthereof.

[0151] The immunoconjugate, antibody fusion protein, naked antibody, andfragments thereof may also be administered to a mammal subcutaneously oreven by other parenteral routes. In a preferred embodiment, theanti-EGP-1 antibody or fragment thereof is administered in a dosage of10 to 2000 milligrams protein per dose, and preferably is repeatedlyadministered. Moreover, the administration may be by continuous infusionor by single or multiple boluses. In general, the dosage of anadministered immunoconjugate, fusion protein or naked antibody forhumans will vary depending upon such factors as the patient's age,weight, height, sex, general medical condition and previous medicalhistory. Typically, it is desirable to provide the recipient with adosage of immunoconjugate, antibody fusion protein or naked antibodythat is in the range of from about 1 mg/kg to 20 mg/kg as a singleintravenous infusion, although a lower or higher dosage also may beadministered as circumstances dictate. This dosage may be repeated asneeded, for example, once per week for 4-10 weeks, preferably once perweek for 8 weeks, and more preferably, once per week for 4 weeks. It mayalso be given less frequently, such as every other week for severalmonths. The dosage may be given through various parenteral routes, withappropriate adjustment of the dose and schedule.

[0152] The RS7 antibodies of the present invention can be formulatedaccording to known methods to prepare pharmaceutically usefulcompositions, whereby RS7 antibodies are combined in a mixture with apharmaceutically acceptable carrier. A composition is said to be a“pharmaceutically acceptable carrier” if its administration can betolerated by a recipient patient. Sterile phosphate-buffered saline isone example of a pharmaceutically acceptable carrier. Other suitablecarriers are well known to those in the art. See, for example,REMINGTON'S PHARMACEUTICAL SCIENCES, 18th Ed. (1990).

[0153] For purposes of therapy, the immunoconjugate, fusion protein, ornaked antibody is administered to a mammal in a therapeuticallyeffective amount. A suitable subject for the present invention isusually a human, although a non-human animal subject is alsocontemplated. An antibody preparation is said to be administered in a“therapeutically effective amount” if the amount administered isphysiologically significant. An agent is physiologically significant ifits presence results in a detectable change in the physiology of arecipient mammal.

[0154] It will be apparent to those skilled in the art that variousmodifications and variations can be made to the compositions andprocesses of this invention. Thus, it is intended that the presentinvention cover such modifications and variations, provided they comewithin the scope of the appended claims and their equivalents.

[0155] The disclosure of all publications, patents and patentapplications cited above are expressly incorporated herein by referencein their entireties to the same extent as if each were incorporated byreference individually.

[0156] The examples below are illustrative of embodiments of the currentinvention and should not be used, in any way, to limit the scope of theclaims.

EXAMPLE 1

[0157] Construction of a Chimeric RS7 Antibody

[0158] Molecular Cloning of RS7Vκ and VH Genes

[0159] Total cytoplasmic RNA and mRNA was prepared from RS7-producinghybridoma cells. The genes encoding Vκ and VH sequences were cloned byRT-PCR and 5′RACE and the sequences were determined by DNA sequencing.Multiple independent clones were sequenced to eliminate possible errorsresulting from the PCR reaction. Sequence analyses revealed presence oftwo Vκ (#1 and #23) and one VH (RS7VH) transcripts. Combining each ofthe putative murine Vκ with the VH, two chimeric Abs (cAbs), containinghuman constant region domains were generated and expressed in Sp2/0cells by transfection. cAb-producing clones were identified by screeningthe cell culture supernatants of the transfected cell clones by ELISA.Positive clones were expanded and cAbs were purified from the cellculture supernatants. The Ag-binding assay showed that only the cAbcomposed of Vκ#23 and VH, cAb-Vκ#23, bound to microwells coated with thecrude membrane fraction of ME180, a human cervical carcinoma cell (ATCC,Rockville, Md.) (FIG. 1). The cAb with the combination of Vκ#1 and VH,cAb-Vκ#1, did not show binding to the Ag-coated wells. Therefore, theimmunoreactive cAb (with Vκ#23) was designated as cRS7. The clonedmurine V_(H) and the functional Vκ (#23) sequences as the final PCRproducts were designated as RS7Vk (FIG. 2A) and RS7VH (FIG. 2B),respectively.

[0160] Binding Activity Assay for RS7 Abs

[0161] A competitive ELISA binding assay was used to evaluate thebinding affinity of engineered cRS7. Briefly, constant amount ofbiotinylated murine RS7 is mixed with varying concentrations (0.01-100μg/ml) of testing Abs (RS7 or cRS7), and added into Ag-coatedmicrowells, and incubated at room temperature for 1 h. After washing,HRP conjugated streptavidin is added and incubated for 1 h at roomtemperature. The amount of HRP-conjugated streptavidin bound to theAg-bound biotinylated RS7 was revealed by reading OD₄₉₀ after theaddition of a substrate solution containing 4 mM ortho-phenylenediaminedihydrochloride and 0.04% H₂O₂. By this type of competitive Ag-bindingassay, it was revealed that cRS7 and murine RS7 competed equally wellfor the binding of biotinylated murine RS7 to the antigen coated wells,thus confirmed the authenticity of the Vκ and VH sequences obtained(FIG. 1).

EXAMPLE 2

[0162] Method of hRS7 Antibody Construction

[0163] Sequence Design of hRS7 V Genes

[0164] By searching the human Vκ and VH sequences in the Kabat database,the FRs of RS7 Vκ and VH were found to exhibit the highest degree ofsequence homology to human SA-1A′cl Vκ and RF-TS3 VH, respectively. Oneexception is the FR4 of RS7VH, which showed the highest sequencehomology with that of NEWM VH. Therefore human SA-1A′CL frameworksequences were used as the scaffold for grafting the CDRs of RS7Vκ (FIG.3A), and a combination of RF-TS3 and NEWM framework sequences were usedfor RS7V_(H) (FIG. 4). There are a number of amino acid changes in eachchain outside of the CDR regions when compared to the starting humanantibody frameworks. Several amino acid residues in murine FRs thatflank the putative CDRs were maintained in the reshaped hRS7 Fv based onthe guideline previously established Qu, Z., Losman, M. J., Eliassen, K.C., Hansen, H. J., Goldenberg, D. M., and Leung, S. O. (1999).Humanization of Immu31, an alpha-fetoprotein-specific antibody. Clin.Cancer Res. 5, 3095s-3100s. These residues are S20, D60, V85, and A100of RS7Vk and K38, K46, A78, and F91 of RS7VH (FIGS. 3A and 3B).

[0165] Construction of hRS7 V Sequences

[0166] A modified strategy as described by Leung et al. Leung, S. O.,Shevitz, J., Pellegrini, M. C., Dion, A. S., Shih, L. B., Goldenberg, D.M., and Hansen, H. J. (1994) Chimerization of LL2, a rapidlyinternalizing antibody specific for B cell lymphoma. Hybridoma, 13:469-476) was used to construct the designed VL and VH genes for hRS7using a combination of long oligonucleotide systheses and PCR asillustrated in FIG. 4. For the construction of the hRS7 VH domain, twolong oligonucleotides, hRS7VHA (176-mer) and hRS7VHB (168-mer) weresynthesized on an automated DNA synthesizer (Applied Biosystem).

[0167] hRS7VHA represents nt 23 to 198 of the hRS7VH domain5′-GGTCTGAGTT GAAGAAGCCT GGGGCCTCAG TGAAGGTTTC CTGCAAGGCT TCTGGATACACCTTCACAAA CTATGGAATG AACTGGGTGA AGCAGGCCCC TGGACAAGGG CTTAAATGGATGGGCTGGAT AAACACCTAC ACTGGAGAGC CAACATATAC TGATGACTTC AAGGGA-3′

[0168] hRS7VHB represents the minus strand of the hRS7VH domaincomplementary to nt 174 to 340. 5′-ACCCTTGGCC CCAGACATCG AAGTACCAGTAGCTACTACC GAACCCCCCT CTTGCACAGA AATACACGGC AGTGTCGTCA GCCTTTAGGCTGCTGATCTG GAGATATGCC GTGCTGACAG AGGTGTCCAA GGAGAAGGCA AACCGTCCCTTGAAGTCATC AGTATATG-3′

[0169] The 3′-terminal sequences (23 nt residues) of hRS7VHA and B arecomplementory to each other. Under defined PCR condition, 3′-ends ofhRS7VHA and B anneal to form a short double stranded DNA flanked by therest of the long oligonucleotides. Each annealed end serves as a primerfor the transcription of the single stranded DNA, resulting in a doublestrand DNA composed of the nt 23 to 340 of hRS7VH. This DNA was furtheramplified in the presence of two short oligonucleotides, hRS7VHBACK andhRS7VHFOR to form the full-length hRS7VH. hRS7VHBACK 5′-GTGGTGCTGCAGCAATCTGG GTCTGAGTTG AAGAAGCC-3′ hRS7VHFOR 5′-TGAGGAGACG GTGACCAGGGACCCTTGGCC CCAGACAT-3′

[0170] Minimum amount of hRS7VHA and B (determined empirically) wasamplified in the presence of 10 μl of 10× PCR Buffer (500 mM KCl, 100 mMTris.HCL buffer, pH 8.3, 15 mM MgCl₂), 2 μmol of hRS7VHBACK andhRS7VHFOR, and 2.5 units of Taq DNA polymerase (Perkin Elmer Cetus,Norwalk, Conn.). This reaction mixture was subjected to 3 cycle of PCRreaction consisting of denaturation at 94° C. for 1 minute, annealing at45° C. for 1 minute, and polymerization at 72° C. for 1.5 minutes, andfollowed by 27 cycles of PCR reaction consisting of denaturation at 94°C. for 1 minute, annealing at 55° C. for 1 minute, and polymerization at72° C. for 1 minute. Double-stranded PCR-amplified product for hRS7VHwas gel-purified, restriction-digested with PstI and BstEII and clonedinto the complementary PstI/BstEII sites of the heavy chain stagingvector, VHpBS2.

[0171] For constructing the full length DNA of the humanized Vκsequence, hRS7VKA (156-mer) and hRS7VKB (155-mer) were synthesized asdescribed above. hRS7VKA and B were amplified by two shortoligonucleotides hRS7VKBACK and hRS7VKFOR as described above.

[0172] HRS7VKA represents nt 20 to 175 of the hRS7Vκ domain.5′-CTCCATCCTC CCTGTCTGCA TCTGTAGGAG ACAGAGTCAG CATCACCTGC AAGGCCAGTCAGGATGTGAG TATTGCTGTA GCCTGGTATC AGCAGAAACC AGGGAAAGCC CCTAAGCTCCTGATCTACTC GGCATCCTAC CGGTACACTG GAGTCC-3′

[0173] hRS7VKB represents the minus strand of the hRS7Vκ domaincomplementary to nt 155 to 320. 5′-CCTTGGTCCC AGCACCGAAC GTGAGCGGAGTAATATAATG TTGCTGACAG TAATAAACTG CAAAATCTTC AGGTTGCAGA CTGCTGATGGTGAGAGTGAA ATCTGTCCCA GATCCACTGC CACTGAACCT ATCAGGGACT CCAGTGTACCGGTAG-3′ hRS7VKBACK 5′-GACATTCAGC TGACCCAGTC TCCATCCTCC CTGTCTG-3′hRS7VKFOR 5′-ACGTTAGATC TCCACCTTGG TCCCAGCACC G-3′

[0174] Gel-purified PCR products for hRS7Vκ were restriction-digestedwith PvuII and BglIII and cloned into the complementary PvuI/BcII sitesof the light chain staging vector, VKpBR2. The final expression vectorhRS7pdHL2 was constructed by sequentially subcloning the XbaI-BamHI andXhoI/BamHI fragments of hRS7Vκ and VH, respectively, into pdHL2 asdescribed above.

[0175] Transfection and Expression of hRS7 Antibodies

[0176] Approximately 30μg of the expression vectors for hRS7werelinearized by digestion with SalI and transfected into Sp2/0-Ag14 cellsby electroporation (450 V and 25 μF). The transfected cells were platedinto 96-well plates for 2 days and then selected for drug-resistance byadding MTX into the medium at a final concentration of 0.025 μM.MTX-resistant colonies emerged in the wells 2-3 weeks. Supernatants fromcolonies surviving selection were screened for human Ab secretion byELISA assay. Briefly, 100 μl supernatants were added into the wells of amicrotiter plate precoated with GAH-IgG, F(ab′)₂ fragment-specific Aband incubated for 1 h at room temperature. Unbound proteins were removedby washing three times with wash buffer (PBS containing 0.05%polysorbate 20). HRP-conjugated GAH-IgG, Fc fragment-specific Ab wasadded to the wells. Following an incubation of 1 h, the plate waswashed. The bound HRP-conjugated Ab was revealed by reading A 490 nmafter the addition of a substrate solution containing 4 mM OPD and 0.04%H₂O₂. Positive cell clones were expanded and hRS7 IgG were purified fromcell culture supernatant by affinity chromatography on a Protein Acolumn.

[0177] Binding Activity of the humanized RS7 antibody

[0178] An ELISA competitive binding assay using ME180 cell membraneextract coated plate was used to assess the immunoreactivity of hRS7 asdescribed (Stein et al., Int. J. Cancer 55:938-946(1993)). ME180 cellmembrane fraction was prepared by sonication and centrifugation. Thecrude membrane extract was coated in 96-well flat bottomed PVC plate bycentrifugation and fixed with 0.1% glutaraldehyde. Constant amount ofthe biotinylated murine RS7 mixed with varying concentrations of mRS7,cRS7 or hRS7 was added to the membrane coated wells and incubated atroom temperature for 1-2 h. After washing, HRP-conjugated streptavidinwas added and incubated for 1 h at room temperature. The amount ofHRP-conjugated streptavidin bound to the membrane-bound biotinylatedmRS7 was revealed by reading A_(490 nm) after the addition of asubstrate solution containing 4 mM ortho-phenylenediaminedihydrochloride and 0.04% H₂O₂. As shown by the competition assays inFIG. 6, hRS7 IgG exhibited comparable binding activities with that ofmRS7 and cRS7, confirming the binding affinity of RS7 was preserved inhumanization.

EXAMPLE 3

[0179] Radioiodinations of Humanized RS7 Using Residualizing Labels

[0180] The residualizing moiety (IMP-R4, IMP-R5 or IMP-R8) wasradioiodinated, and coupled to disulfide-reduced hRS7 along theprocedure described elsewhere (Govindan SV, et al. Bioconjugate Chem.1999;10:231-240). See FIG. 9. In residualizing radioiodine labelingsusing ¹²⁵I, to prepare ¹²⁵I-IMP-Rx-hRS7 where x=4, 5 or 8), overallyields and specific activities (in parentheses) of 87.1% (3.38 mCi/mg),34.3% (0.97 mCi/mg), and 76.6% (2.93 mCi/mg) were obtained using IMP-R4,IMP-R5 and IMP-R8, respectively. In large-scale ¹³¹I labelings using¹³¹I-IMP-R4 entity, the following results were obtained. Using 20.4 mCiof ¹³¹I, 35.7 nmol of IMP-R4 and 3.22 mg of DTT-reduced hRS7, a 60%overall yield (3.80 mCi/mg) was obtained. A different run using 30.3 mCiof ¹³¹I, IMP-R4 and reduced hRS7 produced 69.7% yield (3.88 mCi/mg). Athird run using 13.97 mCi of ¹³¹I gave 71.8% incorporation (4.42mCi/mg). A ¹³¹I-IMP-R4 labeling using 13.6 mCi of ¹³¹I and anon-specific humanized antibody, hLL2, resulted in 64.4% yield (3.67mCi/mg).

EXAMPLE 4

[0181] Preclinical Experiments in Breast Cancer Animal Model

[0182] For tumor targeting studies, tumors were propagated in 5-8 weekold female nude mice by subcutaneous injection of ˜2.3×10⁷ culturedMDA-MB-468 cells, and the animals were used after one month when thetumor size reached ˜01-to-0.2 cm³. The mice were injected i.v. with amixture of ˜10 μCi of ¹²⁵I-[IMP-Rx]-hRS7 where x=4,5 or 8, and 20-25 μCiof ¹³¹I-MAb (CT method). Thus, each experiment was a paired-labelexperiment with ¹²⁵I/¹³¹I. At the indicated times, biodistributions invarious organs and blood were determined, and expressed as % injecteddose per gram. Corrections for backscatter of ¹³¹I into ¹²⁵I window weremade in determining ¹²⁵I biodistributions.

[0183] For therapy studies, tumor growth patterns under various formatswere studied to determine the optimal method for steady growth of tumor.It was concluded that the method used for targeting experiments wasoptimal after about 8-weeks of tumor growth, and 30-50% of the animalscould be used based on the tumor growth profiles. For therapy studies,the tumor-bearing animals were injected i.v. with ¹³¹I-IMPR4-hRS7 wasthe agent examined, and compared with directly radioiodinated material,¹³¹I-hRS7. Baseline body weights were compared with weekly measurementsof body weights and tumor volumes. Animals were sacrificed when tumorsreached 3 cm³. All animal experiments were carried out in accord withIACUC-approved protocols.

[0184] In vivo Animal Biodistributions

[0185] These experiments were carried out using dual-labeled hRS7preparations (¹²⁵I-IMP-Rx-hRS7 where x=4, 5 or 8, with each agent mixedwith direct label ¹³¹I-hRS7) in the tumors grown in NIH Swiss nude mice.Tables 1A, 1B and 1C describe detailed biodistributions showing thesuperior performance using the residualizing labels. For instance, %injected dose per gram of tumor on day-7 were 41.6±3.0%, 32.2±11.6% and24.7±8.5% for ¹²⁵I-IMP-R4-hRS7, ¹²⁵I-IMP-R5-hRS7 and ¹²⁵I-IMP-R8-hRS7,respectively, while that for directly labeled ¹³¹I-hRS7 at the sametime-point in each of the dual-labeled experiments were 5.9±0.9%,6.2±2.1% and 6.7±2.3%. Tumor-to-nontumor ratios for the same time-pointwere 1.7-to-7.6-fold higher with ¹²⁵I-IMP-R4-hRS7, 1.7-to-6.0-foldhigher with ¹²⁵I-IMP-R5-hRS7, and 2.0-to-4.8-fold higher with¹²⁵I-IMP-R8-hRS7 compared to the ratios with 131I-hRS7 (data not shown).

[0186] Table-1. Biodistributions of Humanized RS7, Dual-Labeled with¹²⁵I-IMP-R (R4 or R5 or R8) and ¹³¹I-hRS7 (CT Method), in NIH Swiss NudeMice Bearing MDA-MB-468 Tumor Xenografts TABLE 1A ¹²⁵I-IMP-R4-hRS7versus ¹³¹I-hRS7 (CT) % ID/g ± SD¹, n = 5 Tissue Label 24 h 72 h 168 h,n = 4 336 h MDA- ¹²⁵I-IMP-R4 32.8 ± 6.3  46.8 ± 11.0 41.6 ± 3.0  25.1 ±3.8  MB-468 ¹³¹I (CT) 8.6 ± 1.5 8.6 ± 2.3 5.9 ± 0.9 4.4 ± 0.8 Tumor wt.(0.19 ± 0.06) (0.19 ± 0.08) (0.13 ± 0.07) (0.18 ± 0.04) Liver¹²⁵I-IMP-R4 5.7 ± 0.7 4.7 ± 1.5 2.8 ± 0.4 1.3 ± 0.2 ¹³¹I (CT) 4.1 ± 0.32.0 ± 0.1 1.5 ± 0.2 0.7 ± 0.1 Spleen ¹²⁵I-IMP-R4 3.6 ± 0.6 3.3 ± 0.6 2.6± 0.8 1.9 ± 0.2 ¹³¹I (CT) 2.6 ± 0.5 1.7 ± 0.4 1.1 ± 0.4 0.6 ± 0.1 Kidney¹²⁵I-IMP-R4 7.8 ± 0.7 6.8 ± 0.4 5.6 ± 0.8 3.0 ± 0.5 ¹³¹I (CT) 3.5 ± 0.32.1 ± 0.3 1.4 ± 0.3 0.7 ± 0.1 Lungs ¹²⁵I-IMP-R4 4.5 ± 1.0 3.2 ± 0.6 2.2± 0.7 0.8 ± 0.2 ¹³¹I (CT) 3.1 ± 0.8 2.2 ± 0.4 1.6 ± 0.6 0.6 ± 0.2 Blood¹²⁵I-IMP-R4 15.1 ± 1.4  9.5 ± 0.7 6.0 ± 1.5 1.9 ± 0.6 ¹³¹I (CT) 10.8 ±1.0  7.3 ± 0.6 5.3 ± 1.2 2.2 ± 0.6 Stomach ¹²⁵I-IMP-R4 1.3 ± 0.2 0.6 ±0.1 0.4 ± 0.1 0.2 ± 0.1 ¹³¹I (CT) 1.6 ± 0.5 0.7 ± 0.1 0.4 ± 0.1 0.2 ±0.1 Sm. Int. ¹²⁵I-IMP-R4 1.5 ± 0.2 0.9 ± 0.1 0.6 ± 0.2 0.2 ± 0.1 ¹³¹I(CT) 1.0 ± 0.1 0.6 ± 0.1 0.4 ± 0.1  0.2 ± 0.04 Lg. Int. ¹²⁵I-IMP-R4 1.3± 0.3 1.0 ± 0.1 0.8 ± 0.1 0.3 ± 0.1 ¹³¹I (CT) 0.8 ± 0.2 0.5 ± 0.1 0.5 ±0.1  0.2 ± 0.03 Muscle ¹²⁵I-IMP-R4 1.2 ± 0.2 0.7 ± 0.1 0.5 ± 0.1 0.3 ±0.2 ¹³¹I (CT) 0.9 ± 0.1  0.5 ± 0.05 0.3 ± 0.1 0.2 ± 0.1 Bone ¹²⁵I-IMP-R42.3 ± 0.3 2.1 ± 0.3 2.4 ± 0.6 2.3 ± 1.2 ¹³¹I (CT) 1.4 ± 0.1 0.8 ± 0.10.5 ± 0.1 0.3 ± 0.1

[0187] TABLE 1B ¹²⁵I-IMP-R5-hRS7 versus ¹³¹I-hRS7 (CT method) % ID/g ±SD¹, n = 5 Tissue Label 24 h 72 h 168 h 336 h, n = 4 MDA- ¹²⁵I-IMP-R529.1 ± 4.6  39.6 ± 2.7  32.2 ± 11.6 17.8 ± 7.0  MB-468 ¹³¹I (CT) 9.2 ±1.0 9.1 ± 0.6 6.2 ± 2.1 4.9 ± 2.0 Tumor wt. (0.14 ± 0.02) (0.20 ± 0.05)(0.11 ± 0.03) (0.13 ± 0.06) Liver ¹²⁵I-IMP-R5 4.8 ± 1.4 2.5 ± 0.1 1.8 ±0.3 0.8 ± 0.3 ¹³¹I (CT) 5.1 ± 1.5 2.4 ± 0.2 1.7 ± 0.2 0.8 ± 0.3 Spleen¹²⁵I-IMP-R4 4.1 ± 1.0 2.0 ± 0.4 1.9 ± 0.4 0.8 ± 0.4 ¹³¹I (CT) 3.8 ± 1.21.7 ± 0.5 1.3 ± 0.3 0.7 ± 0.4 Kidney ¹²⁵I-IMP-R4 10.0 ± 1.4  6.3 ± 0.55.0 ± 0.5 1.1 ± 0.3 ¹³¹I (CT) 3.7 ± 0.5 1.9 ± 0.3 1.7 ± 0.3 0.8 ± 0.2Lungs ¹²⁵I-IMP-R4 5.4 ± 1.8 3.2 ± 0.8 2.3 ± 0.2 0.9 ± 0.4 ¹³¹I (CT) 3.9± 1.2 2.5 ± 0.7 2.0 ± 0.3 0.9 ± 0.5 Blood ¹²⁵I-IMP-R4 16.5 ± 4.0  8.8 ±0.6 6.5 ± 1.0 2.7 ± 1.4 ¹³¹I (CT) 12.2 ± 3.0  7.8 ± 0.5 6.3 ± 0.8 3.1 ±1.4 Stomach ¹²⁵I-IMP-R4 0.9 ± 0.2 0.5 ± 0.1 0.4 ± 0.1 0.2 ± 0.1 ¹³¹I(CT) 1.1 ± 0.1 0.6 ± 0.1 0.5 ± 0.1 0.2 ± 0.1 Sm. Int. ¹²⁵I-IMP-R4 1.5 ±0.3  0.8 ± 0.04 0.6 ± 0.1 0.2 ± 0.1 ¹³¹I (CT) 1.1 ± 0.2  0.6 ± 0.02 0.5± 0.1 0.3 ± 0.1 Lg. Int. ¹²⁵I-IMP-R4 1.4 ± 0.2 0.9 ± 0.1 0.6 ± 0.1  0.2± 0.04 ¹³¹I (CT) 0.7 ± 0.1  1.4 ± 0.03 0.4 ± 0.1  0.2 ± 0.04 Muscle¹²⁵I-IMP-R4 1.3 ± 0.3 0.7 ± 0.2 0.5 ± 0.1 0.2 ± 0.1 ¹³¹I (CT) 0.9 ± 0.20.6 ± 0.2 0.4 ± 0.1 0.2 ± 0.1 Bone ¹²⁵I-IMP-R4 2.2 ± 0.6 1.3 ± 0.2 1.2 ±0.5 1.0 ± 0.6 ¹³¹I (CT) 1.9 ± 0.7 0.9 ± 0.1 0.6 ± 0.2 0.3 ± 0.2

[0188] TABLE 1C ¹²⁵I-IMP-R8-hRS7 versus ¹³¹I-hRS7 (CT method) % ID/g ±SD¹, n = 5 Tissue Label 24 h 72 h 168 h 336 h MDA- ¹²⁵I-IMP-R8 24.1 ±5.4  26.9 ± 3.9  24.7 ± 8.5  11.0 ± 6.4  MB-468 ¹³¹I (CT) 8.8 ± 1.6 8.8± 1.0 6.7 ± 2.3 2.4 ± 1.3 Tumor wt. (0.17 ± 0.04) (0.12 ± 0.05) (0.10 ±0.04) (0.15 ± 0.05) Liver ¹²⁵I-IMP-R8 4.6 ± 0.7 3.3 ± 0.4 1.8 ± 0.2 0.7± 0.2 ¹³¹I (CT) 4.1 ± 0.6 3.3 ± 0.4 1.8 ± 0.2 0.8 ± 0.2 Spleen¹²⁵I-IMP-R8 2.6 ± 0.7 2.3 ± 0.2 1.9 ± 0.2 1.0 ± 0.1 ¹³¹I (CT) 2.4 ± 0.82.2 ± 0.3 2.0 ± 0.3 0.7 ± 0.1 Kidney ¹²⁵I-IMP-R8 7.2 ± 0.8 4.6 ± 0.8 2.6± 1.0 1.8 ± 0.1 ¹³¹I (CT) 2.5 ± 0.3 3.0 ± 0.7 1.8 ± 0.5 0.8 ± 0.3 Lungs¹²⁵I-IMP-R8 3.0 ± 0.7 4.7 ± 0.5 2.3 ± 0.6 1.0 ± 0.4 ¹³¹I (CT) 2.4 ± 0.44.4 ± 0.5 2.1 ± 0.5 1.0 ± 0.4 Blood ¹²⁵I-IMP-R8 10.8 ± 1.2  9.6 ± 0.96.3 ± 1.4 2.2 ± 0.6 ¹³¹I (CT) 9.2 ± 1.6 9.5 ± 0.8 6.4 ± 1.4 2.6 ± 0.6Stomach ¹²⁵I-IMP-R8 0.9 ± 0.2 0.7 ± 0.2 0.3 ± 0.1 0.2 ± 0.1 ¹³¹I (CT)1.1 ± 0.2 0.9 ± 0.3 0.4 ± 0.1 0.3 ± 0.1 Sm. Int. ¹²⁵I-IMP-R8 1.0 ± 0.10.8 ± 0.2 0.5 ± 0.1 0.2 ± 0.1 ¹³¹I (CT) 0.8 ± 0.1 0.8 ± 0.1 0.5 ± 0.10.2 ± 0.1 Lg. Int. ¹²⁵I-IMP-R8 1.0 ± 0.1 0.9 ± 0.1 0.5 ± 0.1 0.3 ± 0.1¹³¹I (CT) 0.6 ± 0.1 0.6 ± 0.1 0.4 ± 0.1 0.2 ± 0.1 Muscle ¹²⁵I-IMP-R8 0.8± 0.1 0.6 ± 0.1 0.4 ± 0.1 0.2 ± 0.1 ¹³¹I (CT)  0.6 ± 0.04 0.6 ± 0.1 0.4± 0.1 0.2 ± 0.1 Bone ¹²⁵I-IMP-R8 1.4 ± 0.2 1.2 ± 0.3 1.4 ± 0.2 0.8 ± 0.2¹³¹I (CT) 1.1 ± 0.2 0.9 ± 0.2 0.7 ± 0.1 0.3 ± 0.1

[0189] Dosimetry calculations, based on biodistributions using ¹²⁵I inplace of ¹³¹I, were performed using the method of Siegel, J A andStabin, M G (Journal of Nuclear Medicine 1994; 35:152-156). Table-2compares sets of residualizing and conventional radioiodine labels, andFIG. 10 describes the data graphically. All of the residualizing agentsare seen to perform optimally in terms of dose delivered to tumor andtumor-to-nontumor ratios; ¹³¹I-IMP-R4-hRS7 was chosen for therapyexperiments in view of the advantageous radiochemical yields andspecific activities obtainable for the same agent. TABLE 2 Calculatedradiation doses due to variously radioiodinated hRS7 in the MDA-MB-468tumor model cGy normalized to 1500 cGy to Blood Group I Group II GroupIII Organ Model IMP-R4 CT IMP-R5 CT IMP-R8 CT Tumor (Trap 0 6995 16135187 1506 4000 1206 point 0) Liver Exp 674 456 398 449 497 505 SpleenExp 535 315 336 313 384 356 Kidney Exp 1063 402 867 361 761 394 LungsExp 450 392 450 422 506 473 Blood Exp 1500 1500 1500 1500 1500 1500 (org) Stomach Exp 104 144 84 118 101 128 Sm Int Exp 148 124 131 119 130 121Lg Int Exp 163 108 136 86 140 97 Muscle Exp 112 99 105 100 97 93 BoneExp 486 151 244 149 245 151 mCi for 1500 cGy to blood

[0190] Therapy of MDA-MB-468 Human Breast Carcinoma Xenografts in NudeMice

[0191] Maximum-tolerated-dose (MTD): From dosimetry data (Table-2,group-1), the mCi amounts of ¹³¹I-IMP-R4-hRS7 and ¹³¹I-hRS7, producing aradiation dose of 1500 cGy to blood (estimated MTD) were calculated tobe 0.231 mCi and 0.285 mCi, respectively. Experimental determination ofMTD was carried out using increasing doses of each agent in Swiss nudemice. For ¹³¹I-IMP-R4-hRS7, groups of animals were administered 200,225, 250, 275, 300 and 325 μCi; 1 out of five animals in the 250 μCidose group died by week 4, while 3 out of 4 animals in the 300 μCi dosegroup died between weeks 2 and 4. Although the survival of animals inthe 275 and 325 μCi dose groups at five weeks was unexpected, weconcluded that the MTD was between 231 μCi (calculated from dosimetrydata) and 250 μCi of administered dose. For the ¹³¹I-hRS7 (‘CT’-basedradioiodination), groups of animals were injected with 250, 280, 310,340, 370 and 400 μCi; between weeks 2 and 3, six out of six animals of340 μCi dose group, three out of six animals of 370 μCi dose group, andfour out of four animals of 400 μCi dose group died. Based on these, theMTD was projected to be in the 280-310 μCi range.

[0192] Therapy Study-1

[0193] For this first therapy experiment, comparing the efficacy of¹³¹I-IMPR-4-hRS7 with that of ¹³¹I-hRS7 (CT method), each agent used at˜70% of its maximum-tolerated dose. A single dose of 175 μCi of theresidualizing agent is seen to be significantly more effective than 200μCi of conventional radioiodine agent In this experiment, which alsoincluded untreated controls, 10 or 11 animals were used per group, andall the three groups were randomized such that the distribution ofstarting tumor sizes were very similar. Mean tumor volumes for the threegroups before therapy (day-2) were 0.312±0.181, 0.3081±0.203, and0.303±0.212.

[0194] In this experiment, interim data to day 49 are depicted inFIG.-11 below. The top panel in FIG.11 shows tumor volumes (cm³) forindividual animals in each group, and the bottom panel indicates meantumor volumes in two formats. There were three deaths in the untreatedgroup. Tumor growth control is significantly better for theresidualizing label group compared to the conventional label and theuntreated groups, as determined by the student-t test on the area underthe curves (AUC) for mean tumor volumes (MTV) up to day-49. On day 49,significance (p values) for differences in AUCs of MTVs due to therapywith ¹³¹I-IMP-R4-hRS7, with the respective p values for tumor volumedifferences before therapy (day-2) given in parentheses, are as follows.Versus untreated: 0.05 (0.78); versus 131I-hRS7 (CT): 0.03 (0.98); for¹³¹I-hRS7 (CT) versus untreated: 0.14 (0.81). There is continuingdivergence in mean tumor volumes between the conventional and theresidualizing radioiodine groups on day 49, with the latter groupleading to continued decrease. At 8-weeks post-therapy, completeremissions were observed in 5 of 11 mice treated with ¹³¹I-IMP-R4-hRS7,and the MTV was 20% of the starting value. MTV in the untreated and¹³¹I-hRS7-treated mice at 8 weeks were 280% and 163% of the respectivestarting values, respectively, with 1 complete remission of 11 mice inthe ¹³¹1-hRS7 group.

[0195] The treatments were well tolerated. The mean body weights ofIMP-R4 group on day-2 was 21.93±2.03 and that on day 49 was 23.68±1.81;for ‘CT’ group, the mean body weights were 21.77±2.21 and 23.90±2.64 ondays-2 and 49, respectively. Myelotoxicities of the treated groups, asdetermined by blood cell counts, are shown in FIG. 12. Briefly: With¹³¹I-IMP-R4-hRS7, nadirs of 34%, 7% and 61% of the control levels forWBC, lymphocite and neutrophil counts, respectively, were reached oneweek after the administration of the agent. By week-5, these recoveredto 74%, 58% and 92% of the control levels, respectively, and remained at45%, 36% and 51% of the control levels on day-49; and for ¹³¹I-hRS7(CT): nadirs of 41%, 13% and 67% of the control levels for WBC,lymphocite and neutrophil counts, respectively, were reached one weekafter the administration of the agent. By week-5, these recovered to85%, 67% and 103% of the control levels, respectively, and remained at42%, 32% and 49% of the control levels on day-49.

[0196] Therapy Study-2

[0197] Specificity of RAIT Using ¹³¹I-IMP-R4-hRS7 in the MDA-MB-468Tumor Model

[0198] The efficacy of ¹³¹I-IMP-R4-hRS7 was compared with that ofnon-specific control humanized antibody, hLL2 (anti-CD-22 MAb), labeledwith ¹³¹1-IMP-R4. In this experiment, 175 μCi of each agent wasadministered. This represents ˜70% of the maximum-tolerated dose of¹³¹I-IMP-R4-hRS7. In this experiment, which included untreated controls,7-to-8 animals were used per group, and the groups were randomized withregard to the starting tumor volume distributions as in therapyexperiment-1. FIG. 13, showing the relative mean tumor volumes (MTV) forthe three groups (MTV before therapy: 100) , is indicative of the growthcontrol specificity.

EXAMPLE 5

[0199] Treatment of a Breast Cancer Patient with Y-90 Humanized RS7 mAband with Naked Humanized RS7 mAb

[0200] A 56-year-old women with a history of recurrent adencarcinoma ofthe breast presents with cervical lymph node and left lung metastases.She relapses twice after chemotherapy and hormonal therapies. She isthen given two therapeutic injections, two weeks apart, ofY-90-conjugated humanized RS7 mAb i.v., at a dose each of 20 mCi Y-90 ina protein dose of antibody of 100 mg. Four weeks after therapy, herwhite blood cell and platelet counts have decreased by approximately50%, but recuperate by 9 weeks post-therapy. At the restaging 12 weekspost-therapy, a ca. 30% decrease in pulmonary and nodal metastases hasbeen measured by computed tomography. Thereafter, she receives 4 weeklyinfusions, over 3 hours each, of naked humanized RS7, which is toleratedwell, except for some transient rigors and chills, and without anyadverse effects on her blood counts or blood chemistries. The nakedantibody dose for each infusion was 400 mg/m². Approximately 8 weekslater, restaging by computed tomorgraphy indicates an additionaldecrease in measurable lesions by about 20 percent. At the followupexamination 3 months later, her disease appears to be stable (i.e., noevidence of additional, or progressive growth).

1 36 1 324 DNA Homo sapiens CDS (1)..(324) 1 gac att cag ctg acc cag tctcac aaa ttc atg tcc aca tca gta gga 48 Asp Ile Gln Leu Thr Gln Ser HisLys Phe Met Ser Thr Ser Val Gly 1 5 10 15 gac agg gtc agc atc acc tgcaag gcc agt cag gat gtg agt att gct 96 Asp Arg Val Ser Ile Thr Cys LysAla Ser Gln Asp Val Ser Ile Ala 20 25 30 gta gcc tgg tat caa cag aaa ccagga caa tct cct aaa cta ctg att 144 Val Ala Trp Tyr Gln Gln Lys Pro GlyGln Ser Pro Lys Leu Leu Ile 35 40 45 tac tcg gca tcc tac cgg tac act ggagtc cct gat cgc ttc act ggc 192 Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly ValPro Asp Arg Phe Thr Gly 50 55 60 agt gga tct ggg acg gat ttc act ttc accatc agc agt gtg cag gct 240 Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr IleSer Ser Val Gln Ala 65 70 75 80 gaa gac ctg gca gtt tat tac tgt cag caacat tat att act ccg ctc 288 Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln HisTyr Ile Thr Pro Leu 85 90 95 acg ttc ggt gct ggg acc aag ctg gag ctg aaacgg 324 Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg 100 105 2 108PRT Homo sapiens 2 Asp Ile Gln Leu Thr Gln Ser His Lys Phe Met Ser ThrSer Val Gly 1 5 10 15 Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln AspVal Ser Ile Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser ProLys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro AspArg Phe Thr Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile SerSer Val Gln Ala 65 70 75 80 Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln HisTyr Ile Thr Pro Leu 85 90 95 Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu LysArg 100 105 3 360 DNA Homo sapiens CDS (1)..(360) 3 gtg aag ctg cag gagtca gga cct gag ctg aag aag cct gga gag aca 48 Val Lys Leu Gln Glu SerGly Pro Glu Leu Lys Lys Pro Gly Glu Thr 1 5 10 15 gtc aag atc tcc tgcaag gct tct gga tat acc ttc aca aac tat gga 96 Val Lys Ile Ser Cys LysAla Ser Gly Tyr Thr Phe Thr Asn Tyr Gly 20 25 30 atg aac tgg gtg aag caggct cca gga aag ggt tta aag tgg atg ggc 144 Met Asn Trp Val Lys Gln AlaPro Gly Lys Gly Leu Lys Trp Met Gly 35 40 45 tgg ata aac acc tac act ggagag cca aca tat act gat gac ttc aag 192 Trp Ile Asn Thr Tyr Thr Gly GluPro Thr Tyr Thr Asp Asp Phe Lys 50 55 60 gga cgg ttt gcc ttc tct ttg gaaacc tct gcc acc act gcc tat ttg 240 Gly Arg Phe Ala Phe Ser Leu Glu ThrSer Ala Thr Thr Ala Tyr Leu 65 70 75 80 cag atc aac aac ctc aaa agt gaggac atg gct aca tat ttc tgt gca 288 Gln Ile Asn Asn Leu Lys Ser Glu AspMet Ala Thr Tyr Phe Cys Ala 85 90 95 aga ggg ggg ttc ggt agt agc tac tggtac ttc gat gtc tgg ggc caa 336 Arg Gly Gly Phe Gly Ser Ser Tyr Trp TyrPhe Asp Val Trp Gly Gln 100 105 110 ggg acc acg gtc acc gtc tcc tca 360Gly Thr Thr Val Thr Val Ser Ser 115 120 4 120 PRT Homo sapiens 4 Val LysLeu Gln Glu Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu Thr 1 5 10 15 ValLys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr Gly 20 25 30 MetAsn Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp Met Gly 35 40 45 TrpIle Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Thr Asp Asp Phe Lys 50 55 60 GlyArg Phe Ala Phe Ser Leu Glu Thr Ser Ala Thr Thr Ala Tyr Leu 65 70 75 80Gln Ile Asn Asn Leu Lys Ser Glu Asp Met Ala Thr Tyr Phe Cys Ala 85 90 95Arg Gly Gly Phe Gly Ser Ser Tyr Trp Tyr Phe Asp Val Trp Gly Gln 100 105110 Gly Thr Thr Val Thr Val Ser Ser 115 120 5 106 PRT Homo sapiens 5 AspIle Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 7580 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Leu 85 9095 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile 100 105 6 109 PRT Mus sp. 6Asp Ile Gln Leu Thr Gln Ser His Lys Phe Met Ser Thr Ser Val Gly 1 5 1015 Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Ile Ala 20 2530 Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile 35 4045 Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly 50 5560 Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Val Gln Ala 65 7075 80 Glu Asp Leu Ala Val Thr Tyr Tyr Cys Gln Gln His Tyr Ile Thr Pro 8590 95 Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg 100 105 7 108PRT Homo sapiens 7 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser AlaSer Val Gly 1 5 10 15 Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln AspVal Ser Ile Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala ProLys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro AspArg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile SerSer Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln HisTyr Ile Thr Pro Leu 85 90 95 Thr Phe Gly Ala Gly Thr Lys Val Glu Ile LysArg 100 105 8 117 PRT Homo sapiens 8 Val Gln Leu Val Gln Ser Gly Ser GluLeu Lys Lys Pro Gly Ala Ser 1 5 10 15 Val Lys Val Ser Cys Lys Ala SerGly Tyr Thr Phe Thr Ser Tyr Ala 20 25 30 Met Asn Trp Val Arg Gln Ala ProGly Gln Gly Leu Glu Trp Met Gly 35 40 45 Trp Ile Asn Thr Asn Thr Gly AsnPro Thr Tyr Ala Gln Gly Phe Thr 50 55 60 Gly Arg Phe Val Phe Ser Leu AspThr Ser Val Ser Thr Ala Tyr Leu 65 70 75 80 Gln Ile Ser Ser Leu Lys AlaAsp Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Glu Asp Ser Asn Gly TyrLys Ile Phe Trp Gly Gln Gly Ser Leu 100 105 110 Val Thr Val Ser Ser 1159 120 PRT Mus sp. 9 Val Lys Leu Gln Glu Ser Gly Pro Glu Leu Lys Lys ProGly Glu Thr 1 5 10 15 Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr PheThr Asn Tyr Gly 20 25 30 Met Asn Trp Val Lys Gln Ala Pro Gly Lys Gly LeuLys Trp Met Gly 35 40 45 Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr ThrAsp Asp Phe Lys 50 55 60 Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala ThrThr Ala Tyr Leu 65 70 75 80 Gln Ile Asn Asn Leu Lys Ser Glu Asp Met AlaThr Tyr Phe Cys Ala 85 90 95 Arg Gly Gly Phe Gly Ser Ser Tyr Trp Tyr PheAsp Val Trp Gly Gln 100 105 110 Gly Thr Thr Val Thr Val Ser Ser 115 12010 122 PRT Homo sapiens 10 Gln Val Gln Leu Gln Gln Ser Gly Ser Glu LeuLys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser GlyTyr Thr Phe Thr Asn Tyr 20 25 30 Gly Met Asn Trp Val Lys Gln Ala Pro GlyGln Gly Leu Lys Trp Met 35 40 45 Gly Trp Ile Asn Thr Tyr Thr Gly Glu ProThr Tyr Thr Asp Asp Phe 50 55 60 Lys Gly Arg Phe Ala Phe Ser Leu Asp ThrSer Val Ser Thr Ala Tyr 65 70 75 80 Leu Gln Ile Ser Ser Leu Lys Ala AspAsp Thr Ala Val Tyr Tyr Phe 85 90 95 Cys Ala Arg Gly Gly Phe Gly Ser SerTyr Trp Tyr Phe Asp Val Trp 100 105 110 Gly Gln Gly Ser Leu Val Thr ValSer Ser 115 120 11 324 DNA Homo sapiens CDS (1)..(324) 11 gac atc cagctg acc cag tct cca tcc tcc ctg tct gca tct gta gga 48 Asp Ile Gln LeuThr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 gac aga gtcagc atc acc tgc aag gcc agt cag gat gtg agt att gct 96 Asp Arg Val SerIle Thr Cys Lys Ala Ser Gln Asp Val Ser Ile Ala 20 25 30 gta gcc tgg tatcag cag aaa cca ggg aaa gcc cct aag ctc ctg atc 144 Val Ala Trp Tyr GlnGln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 tac tcg gca tcc taccgg tac act gga gtc cct gat agg ttc agt ggc 192 Tyr Ser Ala Ser Tyr ArgTyr Thr Gly Val Pro Asp Arg Phe Ser Gly 50 55 60 agt gga tct ggg aca gatttc act ctc acc atc agc agt ctg caa cct 240 Ser Gly Ser Gly Thr Asp PheThr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 gaa gat ttt gca gtt tattac tgt cag caa cat tat att act ccg ctc 288 Glu Asp Phe Ala Val Tyr TyrCys Gln Gln His Tyr Ile Thr Pro Leu 85 90 95 acg ttc ggt gct ggg acc aaggtg gag atc aaa cgt 324 Thr Phe Gly Ala Gly Thr Lys Val Glu Ile Lys Arg100 105 12 108 PRT Homo sapiens 12 Asp Ile Gln Leu Thr Gln Ser Pro SerSer Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Ser Ile Thr Cys LysAla Ser Gln Asp Val Ser Ile Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys ProGly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Tyr Arg Tyr ThrGly Val Pro Asp Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe ThrLeu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr TyrCys Gln Gln His Tyr Ile Thr Pro Leu 85 90 95 Thr Phe Gly Ala Gly Thr LysVal Glu Ile Lys Arg 100 105 13 363 DNA Homo sapiens CDS (1)..(363) 13cag gtc caa ctg cag caa tct ggg tct gag ttg aag aag cct ggg gcc 48 GlnVal Gln Leu Gln Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala 1 5 10 15tca gtg aag gtt tcc tgc aag gct tct gga tac acc ttc aca aac tat 96 SerVal Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 ggaatg aac tgg gtg aag cag gcc cct gga caa ggg ctt aaa tgg atg 144 Gly MetAsn Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Lys Trp Met 35 40 45 ggc tggata aac acc tac act gga gag cca aca tat act gat gac ttc 192 Gly Trp IleAsn Thr Tyr Thr Gly Glu Pro Thr Tyr Thr Asp Asp Phe 50 55 60 aag gga cggttt gcc ttc tcc ttg gac acc tct gtc agc acg gca tat 240 Lys Gly Arg PheAla Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr 65 70 75 80 ctc cag atcagc agc cta aag gct gac gac act gcc gtg tat ttc tgt 288 Leu Gln Ile SerSer Leu Lys Ala Asp Asp Thr Ala Val Tyr Phe Cys 85 90 95 gca aga ggg gggttc ggt agt agc tac tgg tac ttc gat gtc tgg ggc 336 Ala Arg Gly Gly PheGly Ser Ser Tyr Trp Tyr Phe Asp Val Trp Gly 100 105 110 caa ggg tcc ctggtc acc gtc tcc tca 363 Gln Gly Ser Leu Val Thr Val Ser Ser 115 120 14121 PRT Homo sapiens 14 Gln Val Gln Leu Gln Gln Ser Gly Ser Glu Leu LysLys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly TyrThr Phe Thr Asn Tyr 20 25 30 Gly Met Asn Trp Val Lys Gln Ala Pro Gly GlnGly Leu Lys Trp Met 35 40 45 Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro ThrTyr Thr Asp Asp Phe 50 55 60 Lys Gly Arg Phe Ala Phe Ser Leu Asp Thr SerVal Ser Thr Ala Tyr 65 70 75 80 Leu Gln Ile Ser Ser Leu Lys Ala Asp AspThr Ala Val Tyr Phe Cys 85 90 95 Ala Arg Gly Gly Phe Gly Ser Ser Tyr TrpTyr Phe Asp Val Trp Gly 100 105 110 Gln Gly Ser Leu Val Thr Val Ser Ser115 120 15 702 DNA Homo sapiens CDS (1)..(699) 15 atg gga tgg agc tgtatc atc ctc ttc ttg gta gca aca gct aca ggt 48 Met Gly Trp Ser Cys IleIle Leu Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 gtc cac tcc gac atccag ctg acc cag tct cca tcc tcc ctg tct gca 96 Val His Ser Asp Ile GlnLeu Thr Gln Ser Pro Ser Ser Leu Ser Ala 20 25 30 tct gta gga gac aga gtcagc atc acc tgc aag gcc agt cag gat gtg 144 Ser Val Gly Asp Arg Val SerIle Thr Cys Lys Ala Ser Gln Asp Val 35 40 45 agt att gct gta gcc tgg tatcag cag aaa cca ggg aaa gcc cct aag 192 Ser Ile Ala Val Ala Trp Tyr GlnGln Lys Pro Gly Lys Ala Pro Lys 50 55 60 ctc ctg atc tac tcg gca tcc taccgg tac act gga gtc cct gat agg 240 Leu Leu Ile Tyr Ser Ala Ser Tyr ArgTyr Thr Gly Val Pro Asp Arg 65 70 75 80 ttc agt ggc agt gga tct ggg acagat ttc act ctc acc atc agc agt 288 Phe Ser Gly Ser Gly Ser Gly Thr AspPhe Thr Leu Thr Ile Ser Ser 85 90 95 ctg caa cct gaa gat ttt gca gtt tattac tgt cag caa cat tat att 336 Leu Gln Pro Glu Asp Phe Ala Val Tyr TyrCys Gln Gln His Tyr Ile 100 105 110 act ccg ctc acg ttc ggt gct ggg accaag gtg gag atc aaa cgt act 384 Thr Pro Leu Thr Phe Gly Ala Gly Thr LysVal Glu Ile Lys Arg Thr 115 120 125 gtg gct gca cca tct gtc ttc atc ttcccg cca tct gat gag cag ttg 432 Val Ala Ala Pro Ser Val Phe Ile Phe ProPro Ser Asp Glu Gln Leu 130 135 140 aaa tct gga act gcc tct gtt gtg tgcctg ctg aat aac ttc tat ccc 480 Lys Ser Gly Thr Ala Ser Val Val Cys LeuLeu Asn Asn Phe Tyr Pro 145 150 155 160 aga gag gcc aaa gta cag tgg aaggtg gat aac gcc ctc caa tcg ggt 528 Arg Glu Ala Lys Val Gln Trp Lys ValAsp Asn Ala Leu Gln Ser Gly 165 170 175 aac tcc cag gag agt gtc aca gagcag gac agc aag gac agc acc tac 576 Asn Ser Gln Glu Ser Val Thr Glu GlnAsp Ser Lys Asp Ser Thr Tyr 180 185 190 agc ctc agc agc acc ctg acg ctgagc aaa gca gac tac gag aaa cac 624 Ser Leu Ser Ser Thr Leu Thr Leu SerLys Ala Asp Tyr Glu Lys His 195 200 205 aaa gtc tac gcc tgc gaa gtc acccat cag ggc ctg agc tcg ccc gtc 672 Lys Val Tyr Ala Cys Glu Val Thr HisGln Gly Leu Ser Ser Pro Val 210 215 220 aca aag agc ttc aac agg gga gagtgt tag 702 Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 16 233 PRT Homosapiens 16 Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala ThrGly 1 5 10 15 Val His Ser Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser LeuSer Ala 20 25 30 Ser Val Gly Asp Arg Val Ser Ile Thr Cys Lys Ala Ser GlnAsp Val 35 40 45 Ser Ile Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys AlaPro Lys 50 55 60 Leu Leu Ile Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val ProAsp Arg 65 70 75 80 Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu ThrIle Ser Ser 85 90 95 Leu Gln Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln GlnHis Tyr Ile 100 105 110 Thr Pro Leu Thr Phe Gly Ala Gly Thr Lys Val GluIle Lys Arg Thr 115 120 125 Val Ala Ala Pro Ser Val Phe Ile Phe Pro ProSer Asp Glu Gln Leu 130 135 140 Lys Ser Gly Thr Ala Ser Val Val Cys LeuLeu Asn Asn Phe Tyr Pro 145 150 155 160 Arg Glu Ala Lys Val Gln Trp LysVal Asp Asn Ala Leu Gln Ser Gly 165 170 175 Asn Ser Gln Glu Ser Val ThrGlu Gln Asp Ser Lys Asp Ser Thr Tyr 180 185 190 Ser Leu Ser Ser Thr LeuThr Leu Ser Lys Ala Asp Tyr Glu Lys His 195 200 205 Lys Val Tyr Ala CysGlu Val Thr His Gln Gly Leu Ser Ser Pro Val 210 215 220 Thr Lys Ser PheAsn Arg Gly Glu Cys 225 230 17 1410 DNA Homo sapiens CDS (1)..(1407) 17atg gga tgg agc tgt atc atc ctc ttc ttg gta gca aca gct aca ggt 48 MetGly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15gtc cac tcc gtc caa ctg cag caa tct ggg tct gag ttg aag aag cct 96 ValHis Ser Val Gln Leu Gln Gln Ser Gly Ser Glu Leu Lys Lys Pro 20 25 30 ggggcc tca gtg aag gtt tcc tgc aag gct tct gga tac acc ttc aca 144 Gly AlaSer Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr 35 40 45 aac tatgga atg aac tgg gtg aag cag gcc cct gga caa ggg ctt aaa 192 Asn Tyr GlyMet Asn Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Lys 50 55 60 tgg atg ggctgg ata aac acc tac act gga gag cca aca tat act gat 240 Trp Met Gly TrpIle Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Thr Asp 65 70 75 80 gac ttc aaggga cgg ttt gcc ttc tcc ttg gac acc tct gtc agc acg 288 Asp Phe Lys GlyArg Phe Ala Phe Ser Leu Asp Thr Ser Val Ser Thr 85 90 95 gca tat ctc cagatc agc agc cta aag gct gac gac act gcc gtg tat 336 Ala Tyr Leu Gln IleSer Ser Leu Lys Ala Asp Asp Thr Ala Val Tyr 100 105 110 ttc tgt gca agaggg ggg ttc ggt agt agc tac tgg tac ttc gat gtc 384 Phe Cys Ala Arg GlyGly Phe Gly Ser Ser Tyr Trp Tyr Phe Asp Val 115 120 125 tgg ggc caa gggtcc ctg gtc acc gtc tcc tca gcc tcc acc aag ggc 432 Trp Gly Gln Gly SerLeu Val Thr Val Ser Ser Ala Ser Thr Lys Gly 130 135 140 cca tcg gtc ttcccc ctg gca ccc tcc tcc aag agc acc tct ggg ggc 480 Pro Ser Val Phe ProLeu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly 145 150 155 160 aca gcg gccctg ggc tgc ctg gtc aag gac tac ttc ccc gaa ccg gtg 528 Thr Ala Ala LeuGly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 165 170 175 acg gtg tcgtgg aac tca ggc gcc ctg acc agc ggc gtg cac acc ttc 576 Thr Val Ser TrpAsn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe 180 185 190 ccg gct gtccta cag tcc tca gga ctc tac tcc ctc agc agc gtg gtg 624 Pro Ala Val LeuGln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 195 200 205 acc gtg ccctcc agc agc ttg ggc acc cag acc tac atc tgc aac gtg 672 Thr Val Pro SerSer Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val 210 215 220 aat cac aagccc agc aac acc aag gtg gac aag aga gtt gag ccc aaa 720 Asn His Lys ProSer Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys 225 230 235 240 tct tgtgac aaa act cac aca tgc cca ccg tgc cca gca cct gaa ctc 768 Ser Cys AspLys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu 245 250 255 ctg ggggga ccg tca gtc ttc ctc ttc ccc cca aaa ccc aag gac acc 816 Leu Gly GlyPro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 260 265 270 ctc atgatc tcc cgg acc cct gag gtc aca tgc gtg gtg gtg gac gtg 864 Leu Met IleSer Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 275 280 285 agc cacgaa gac cct gag gtc aag ttc aac tgg tac gtg gac ggc gtg 912 Ser His GluAsp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 290 295 300 gag gtgcat aat gcc aag aca aag ccg cgg gag gag cag tac aac agc 960 Glu Val HisAsn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 305 310 315 320 acgtac cgt gtg gtc agc gtc ctc acc gtc ctg cac cag gac tgg ctg 1008 Thr TyrArg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 325 330 335 aatggc aag gag tac aag tgc aag gtc tcc aac aaa gcc ctc cca gcc 1056 Asn GlyLys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 340 345 350 cccatc gag aaa acc atc tcc aaa gcc aaa ggg cag ccc cga gaa cca 1104 Pro IleGlu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 355 360 365 caggtg tac acc ctg ccc cca tcc cgg gag gag atg acc aag aac cag 1152 Gln ValTyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln 370 375 380 gtcagc ctg acc tgc ctg gtc aaa ggc ttc tat ccc agc gac atc gcc 1200 Val SerLeu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 385 390 395 400gtg gag tgg gag agc aat ggg cag ccg gag aac aac tac aag acc acg 1248 ValGlu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 405 410 415cct ccc gtg ctg gac tcc gac ggc tcc ttc ttc ctc tat agc aag ctc 1296 ProPro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 420 425 430acc gtg gac aag agc agg tgg cag cag ggg aac gtc ttc tca tgc tcc 1344 ThrVal Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 435 440 445gtg atg cat gag gct ctg cac aac cac tac acg cag aag agc ctc tcc 1392 ValMet His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 450 455 460ctg tct ccg ggt aaa tga 1410 Leu Ser Pro Gly Lys 465 18 469 PRT Homosapiens 18 Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala ThrGly 1 5 10 15 Val His Ser Val Gln Leu Gln Gln Ser Gly Ser Glu Leu LysLys Pro 20 25 30 Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr ThrPhe Thr 35 40 45 Asn Tyr Gly Met Asn Trp Val Lys Gln Ala Pro Gly Gln GlyLeu Lys 50 55 60 Trp Met Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr TyrThr Asp 65 70 75 80 Asp Phe Lys Gly Arg Phe Ala Phe Ser Leu Asp Thr SerVal Ser Thr 85 90 95 Ala Tyr Leu Gln Ile Ser Ser Leu Lys Ala Asp Asp ThrAla Val Tyr 100 105 110 Phe Cys Ala Arg Gly Gly Phe Gly Ser Ser Tyr TrpTyr Phe Asp Val 115 120 125 Trp Gly Gln Gly Ser Leu Val Thr Val Ser SerAla Ser Thr Lys Gly 130 135 140 Pro Ser Val Phe Pro Leu Ala Pro Ser SerLys Ser Thr Ser Gly Gly 145 150 155 160 Thr Ala Ala Leu Gly Cys Leu ValLys Asp Tyr Phe Pro Glu Pro Val 165 170 175 Thr Val Ser Trp Asn Ser GlyAla Leu Thr Ser Gly Val His Thr Phe 180 185 190 Pro Ala Val Leu Gln SerSer Gly Leu Tyr Ser Leu Ser Ser Val Val 195 200 205 Thr Val Pro Ser SerSer Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val 210 215 220 Asn His Lys ProSer Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys 225 230 235 240 Ser CysAsp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu 245 250 255 LeuGly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 260 265 270Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 275 280285 Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 290295 300 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser305 310 315 320 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln AspTrp Leu 325 330 335 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys AlaLeu Pro Ala 340 345 350 Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly GlnPro Arg Glu Pro 355 360 365 Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu GluMet Thr Lys Asn Gln 370 375 380 Val Ser Leu Thr Cys Leu Val Lys Gly PheTyr Pro Ser Asp Ile Ala 385 390 395 400 Val Glu Trp Glu Ser Asn Gly GlnPro Glu Asn Asn Tyr Lys Thr Thr 405 410 415 Pro Pro Val Leu Asp Ser AspGly Ser Phe Phe Leu Tyr Ser Lys Leu 420 425 430 Thr Val Asp Lys Ser ArgTrp Gln Gln Gly Asn Val Phe Ser Cys Ser 435 440 445 Val Met His Glu AlaLeu His Asn His Tyr Thr Gln Lys Ser Leu Ser 450 455 460 Leu Ser Pro GlyLys 465 19 702 DNA Homo sapiens CDS (1)..(699) 19 atg gga tgg agc tgtatc atc ctc ttc ttg gta gca aca gct aca ggt 48 Met Gly Trp Ser Cys IleIle Leu Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 gtc cac tcc gac atccag ctg acc cag tct cca tcc tcc ctg tct gca 96 Val His Ser Asp Ile GlnLeu Thr Gln Ser Pro Ser Ser Leu Ser Ala 20 25 30 tct gta gga gac aga gtcagc atc acc tgc aag gcc agt cag gat gtg 144 Ser Val Gly Asp Arg Val SerIle Thr Cys Lys Ala Ser Gln Asp Val 35 40 45 agt att gct gta gcc tgg tatcag cag aaa cca ggg aaa gcc cct aag 192 Ser Ile Ala Val Ala Trp Tyr GlnGln Lys Pro Gly Lys Ala Pro Lys 50 55 60 ctc ctg atc tac tcg gca tcc taccgg tac act gga gtc cct gat agg 240 Leu Leu Ile Tyr Ser Ala Ser Tyr ArgTyr Thr Gly Val Pro Asp Arg 65 70 75 80 ttc agt ggc agt gga tct ggg acagat ttc act ctc acc atc agc agt 288 Phe Ser Gly Ser Gly Ser Gly Thr AspPhe Thr Leu Thr Ile Ser Ser 85 90 95 ctg caa cct gaa gat ttt gca gtt tattac tgt cag caa cat tat att 336 Leu Gln Pro Glu Asp Phe Ala Val Tyr TyrCys Gln Gln His Tyr Ile 100 105 110 act ccg ctc acg ttc ggt gct ggg accaag gtg gag atc aaa cgt act 384 Thr Pro Leu Thr Phe Gly Ala Gly Thr LysVal Glu Ile Lys Arg Thr 115 120 125 gtg gct gca cca tct gtc ttc atc ttcccg cca tct gat gag cag ttg 432 Val Ala Ala Pro Ser Val Phe Ile Phe ProPro Ser Asp Glu Gln Leu 130 135 140 aaa tct gga act gcc tct gtt gtg tgcctg ctg aat aac ttc tat ccc 480 Lys Ser Gly Thr Ala Ser Val Val Cys LeuLeu Asn Asn Phe Tyr Pro 145 150 155 160 aga gag gcc aaa gta cag tgg aaggtg gat aac gcc ctc caa tcg ggt 528 Arg Glu Ala Lys Val Gln Trp Lys ValAsp Asn Ala Leu Gln Ser Gly 165 170 175 aac tcc cag gag agt gtc aca gagcag gac agc aag gac agc acc tac 576 Asn Ser Gln Glu Ser Val Thr Glu GlnAsp Ser Lys Asp Ser Thr Tyr 180 185 190 agc ctc agc agc acc ctg acg ctgagc aaa gca gac tac gag aaa cac 624 Ser Leu Ser Ser Thr Leu Thr Leu SerLys Ala Asp Tyr Glu Lys His 195 200 205 aaa gtc tac gcc tgc gaa gtc acccat cag ggc ctg agc tcg ccc gtc 672 Lys Val Tyr Ala Cys Glu Val Thr HisGln Gly Leu Ser Ser Pro Val 210 215 220 aca aag agc ttc aac agg gga gagtgt tag 702 Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 20 233 PRT Homosapiens 20 Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala ThrGly 1 5 10 15 Val His Ser Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser LeuSer Ala 20 25 30 Ser Val Gly Asp Arg Val Ser Ile Thr Cys Lys Ala Ser GlnAsp Val 35 40 45 Ser Ile Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys AlaPro Lys 50 55 60 Leu Leu Ile Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val ProAsp Arg 65 70 75 80 Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu ThrIle Ser Ser 85 90 95 Leu Gln Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln GlnHis Tyr Ile 100 105 110 Thr Pro Leu Thr Phe Gly Ala Gly Thr Lys Val GluIle Lys Arg Thr 115 120 125 Val Ala Ala Pro Ser Val Phe Ile Phe Pro ProSer Asp Glu Gln Leu 130 135 140 Lys Ser Gly Thr Ala Ser Val Val Cys LeuLeu Asn Asn Phe Tyr Pro 145 150 155 160 Arg Glu Ala Lys Val Gln Trp LysVal Asp Asn Ala Leu Gln Ser Gly 165 170 175 Asn Ser Gln Glu Ser Val ThrGlu Gln Asp Ser Lys Asp Ser Thr Tyr 180 185 190 Ser Leu Ser Ser Thr LeuThr Leu Ser Lys Ala Asp Tyr Glu Lys His 195 200 205 Lys Val Tyr Ala CysGlu Val Thr His Gln Gly Leu Ser Ser Pro Val 210 215 220 Thr Lys Ser PheAsn Arg Gly Glu Cys 225 230 21 1410 DNA Homo sapiens CDS (1)..(1407) 21atg gga tgg agc tgt atc atc ctc ttc ttg gta gca aca gct aca ggt 48 MetGly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15gtc cac tcc gtc caa ctg cag caa tct ggg tct gag ttg aag aag cct 96 ValHis Ser Val Gln Leu Gln Gln Ser Gly Ser Glu Leu Lys Lys Pro 20 25 30 gggccc tca gtg aag gtt tcc tgc aag gct tct gga tac acc ttc aca 144 Gly ProSer Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr 35 40 45 aac tatgga atg aac tgg gtg aag cag gcc cct gga caa ggg ctt aaa 192 Asn Tyr GlyMet Asn Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Lys 50 55 60 tgg atg ggctgg ata aac acc tac act gga gag cca aca tat act gat 240 Trp Met Gly TrpIle Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Thr Asp 65 70 75 80 gac ttc aaggga cgg ttt gcc ttc tcc ttg gac acc tct gtc agc acg 288 Asp Phe Lys GlyArg Phe Ala Phe Ser Leu Asp Thr Ser Val Ser Thr 85 90 95 gca tat ctc cagatc agc agc cta aag gct gac gac act gcc gtg tat 336 Ala Tyr Leu Gln IleSer Ser Leu Lys Ala Asp Asp Thr Ala Val Tyr 100 105 110 ttc tgt gca agaggg ggg ttc ggt agt agc tac tgg tac ttc gat gtc 384 Phe Cys Ala Arg GlyGly Phe Gly Ser Ser Tyr Trp Tyr Phe Asp Val 115 120 125 tgg ggc caa gggtcc ctg gtc acc gtc tcc tca gcc tcc acc aag ggc 432 Trp Gly Gln Gly SerLeu Val Thr Val Ser Ser Ala Ser Thr Lys Gly 130 135 140 cca tcg gtc ttcccc ctg gca ccc tcc tcc aag agc acc tct ggg ggc 480 Pro Ser Val Phe ProLeu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly 145 150 155 160 aca gcg gccctg ggc tgc ctg gtc aag gac tac ttc ccc gaa ccg gtg 528 Thr Ala Ala LeuGly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 165 170 175 acg gtg tcgtgg aac tca ggc gcc ctg acc agc ggc gtg cac acc ttc 576 Thr Val Ser TrpAsn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe 180 185 190 ccg gct gtccta cag tcc tca gga ctc tac tcc ctc agc agc gtg gtg 624 Pro Ala Val LeuGln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 195 200 205 acc gtg ccctcc agc agc ttg ggc acc cag acc tac atc tgc aac gtg 672 Thr Val Pro SerSer Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val 210 215 220 aat cac aagccc agc aac acc aag gtg gac aag aga gtt gag ccc aaa 720 Asn His Lys ProSer Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys 225 230 235 240 tct tgtgac aaa act cac aca tgc cca ccg tgc cca gca cct gaa ctc 768 Ser Cys AspLys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu 245 250 255 ctg ggggga ccg tca gtc ttc ctc ttc ccc cca aaa ccc aag gac acc 816 Leu Gly GlyPro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 260 265 270 ctc atgatc tcc cgg acc cct gag gtc aca tgc gtg gtg gtg gac gtg 864 Leu Met IleSer Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 275 280 285 agc cacgaa gac cct gag gtc aag ttc aac tgg tac gtg gac ggc gtg 912 Ser His GluAsp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 290 295 300 gag gtgcat aat gcc aag aca aag ccg cgg gag gag cag tac aac agc 960 Glu Val HisAsn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 305 310 315 320 acgtac cgt gtg gtc agc gtc ctc acc gtc ctg cac cag gac tgg ctg 1008 Thr TyrArg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 325 330 335 aatggc aag gag tac aag tgc aag gtc tcc aac aaa gcc ctc cca gcc 1056 Asn GlyLys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 340 345 350 cccatc gag aaa acc atc tcc aaa gcc aaa ggg cag ccc cga gaa cca 1104 Pro IleGlu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 355 360 365 caggtg tac acc ctg ccc cca tcc cgg gag gag atg acc aag aac cag 1152 Gln ValTyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln 370 375 380 gtcagc ctg acc tgc ctg gtc aaa ggc ttc tat ccc agc gac atc gcc 1200 Val SerLeu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 385 390 395 400gtg gag tgg gag agc aat ggg cag ccg gag aac aac tac aag acc acg 1248 ValGlu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 405 410 415cct ccc gtg ctg gac tcc gac ggc tcc ttc ttc ctc tat agc aag ctc 1296 ProPro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 420 425 430acc gtg gac aag agc agg tgg cag cag ggg aac gtc ttc tca tgc tcc 1344 ThrVal Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 435 440 445gtg atg cat gag gct ctg cac aac cac tac acg cag aag agc ctc tcc 1392 ValMet His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 450 455 460ctg tct ccg ggt aaa tga 1410 Leu Ser Pro Gly Lys 465 22 469 PRT Homosapiens 22 Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala ThrGly 1 5 10 15 Val His Ser Val Gln Leu Gln Gln Ser Gly Ser Glu Leu LysLys Pro 20 25 30 Gly Pro Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr ThrPhe Thr 35 40 45 Asn Tyr Gly Met Asn Trp Val Lys Gln Ala Pro Gly Gln GlyLeu Lys 50 55 60 Trp Met Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr TyrThr Asp 65 70 75 80 Asp Phe Lys Gly Arg Phe Ala Phe Ser Leu Asp Thr SerVal Ser Thr 85 90 95 Ala Tyr Leu Gln Ile Ser Ser Leu Lys Ala Asp Asp ThrAla Val Tyr 100 105 110 Phe Cys Ala Arg Gly Gly Phe Gly Ser Ser Tyr TrpTyr Phe Asp Val 115 120 125 Trp Gly Gln Gly Ser Leu Val Thr Val Ser SerAla Ser Thr Lys Gly 130 135 140 Pro Ser Val Phe Pro Leu Ala Pro Ser SerLys Ser Thr Ser Gly Gly 145 150 155 160 Thr Ala Ala Leu Gly Cys Leu ValLys Asp Tyr Phe Pro Glu Pro Val 165 170 175 Thr Val Ser Trp Asn Ser GlyAla Leu Thr Ser Gly Val His Thr Phe 180 185 190 Pro Ala Val Leu Gln SerSer Gly Leu Tyr Ser Leu Ser Ser Val Val 195 200 205 Thr Val Pro Ser SerSer Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val 210 215 220 Asn His Lys ProSer Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys 225 230 235 240 Ser CysAsp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu 245 250 255 LeuGly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 260 265 270Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 275 280285 Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 290295 300 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser305 310 315 320 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln AspTrp Leu 325 330 335 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys AlaLeu Pro Ala 340 345 350 Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly GlnPro Arg Glu Pro 355 360 365 Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu GluMet Thr Lys Asn Gln 370 375 380 Val Ser Leu Thr Cys Leu Val Lys Gly PheTyr Pro Ser Asp Ile Ala 385 390 395 400 Val Glu Trp Glu Ser Asn Gly GlnPro Glu Asn Asn Tyr Lys Thr Thr 405 410 415 Pro Pro Val Leu Asp Ser AspGly Ser Phe Phe Leu Tyr Ser Lys Leu 420 425 430 Thr Val Asp Lys Ser ArgTrp Gln Gln Gly Asn Val Phe Ser Cys Ser 435 440 445 Val Met His Glu AlaLeu His Asn His Tyr Thr Gln Lys Ser Leu Ser 450 455 460 Leu Ser Pro GlyLys 465 23 11 PRT Mus sp. 23 Lys Ala Ser Gln Asp Val Ser Ile Ala Val Ala1 5 10 24 7 PRT Mus sp. 24 Ser Ala Ser Tyr Arg Tyr Thr 1 5 25 9 PRT Mussp. 25 Gln Gln His Tyr Ile Thr Pro Leu Thr 1 5 26 5 PRT Mus sp. 26 AsnTyr Gly Met Asn 1 5 27 17 PRT Mus sp. 27 Trp Ile Asn Thr Tyr Thr Gly GluPro Thr Tyr Thr Asp Asp Phe Lys 1 5 10 15 Gly 28 12 PRT Mus sp. 28 GlyGly Phe Gly Ser Ser Tyr Trp Tyr Phe Asp Val 1 5 10 29 176 DNA ArtificialSequence Description of Artificial Sequence Synthetic oligonucleotide 29ggtctgagtt gaagaagcct ggggcctcag tgaaggtttc ctgcaaggct tctggataca 60ccttcacaaa ctatggaatg aactgggtga agcaggcccc tggacaaggg cttaaatgga 120tgggctggat aaacacctac actggagagc caacatatac tgatgacttc aaggga 176 30 168DNA Artificial Sequence Description of Artificial Sequence Syntheticoligonucleotide 30 acccttggcc ccagacatcg aagtaccagt agctactaccgaacccccct cttgcacaga 60 aatacacggc agtgtcgtca gcctttaggc tgctgatctggagatatgcc gtgctgacag 120 aggtgtccaa ggagaaggca aaccgtccct tgaagtcatcagtatatg 168 31 38 DNA Artificial Sequence Description of ArtificialSequence Synthetic oligonucleotide 31 gtggtgctgc agcaatctgg gtctgagttgaagaagcc 38 32 38 DNA Artificial Sequence Description of ArtificialSequence Synthetic oligonucleotide 32 tgaggagacg gtgaccaggg acccttggccccagacat 38 33 156 DNA Artificial Sequence Description of ArtificialSequence Synthetic oligonucleotide 33 ctccatcctc cctgtctgca tctgtaggagacagagtcag catcacctgc aaggccagtc 60 aggatgtgag tattgctgta gcctggtatcagcagaaacc agggaaagcc cctaagctcc 120 tgatctactc ggcatcctac cggtacactggagtcc 156 34 155 DNA Artificial Sequence Description of ArtificialSequence Synthetic oligonucleotide 34 ccttggtccc agcaccgaac gtgagcggagtaatataatg ttgctgacag taataaactg 60 caaaatcttc aggttgcaga ctgctgatggtgagagtgaa atctgtccca gatccactgc 120 cactgaacct atcagggact ccagtgtaccggtag 155 35 37 DNA Artificial Sequence Description of ArtificialSequence Synthetic oligonucleotide 35 gacattcagc tgacccagtc tccatcctccctgtctg 37 36 31 DNA Artificial Sequence Description of ArtificialSequence Synthetic oligonucleotide 36 acgttagatc tccaccttgg tcccagcacc g31

What is claimed:
 1. An antibody or fragment thereof that binds to anEGP-1 glycoprotein.
 2. The antibody or fragment thereof of claim 1,wherein said antibody or fragment thereof is an RS7 antibody or fragmentthereof.
 3. The antibody or fragment thereof of claim 2, wherein saidantibody or fragment thereof is humanized.
 4. The antibody or fragmentthereof of claim 2, wherein said antibody or fragment thereof is fullyhuman.
 5. The humanized antibody or fragment thereof of claim 3, whereinthe complementarity determining regions (CDRs) of the light chainvariable region of the humanized RS7 MAb comprises CDR1 comprising anamino acid sequence of KASQDVSIAVA; CDR2 comprising an amino acidsequence of SASYRYT; and CDR3 comprising an amino acid sequence ofQQHYITPLT.
 6. The humanized antibody or fragment thereof of claim 3,wherein the CDRs of the heavy chain variable region of the humanized RS7MAb comprises CDR1 comprising an amino acid sequence of NYGMN; CDR2comprising an amino acid sequence of WINTYTGEPTYTDDFKG and CDR3comprising an amino acid sequence of GGFGSSYWYFDV.
 7. The humanizedantibody or fragment thereof of claim 3, comprising the CDRs of a murineRS7 MAb and the framework region (FR) of the light and heavy chainvariable regions of a human antibody, wherein the CDRs of the lightchain variable region of the humanized RS7 MAb comprises CDR1 comprisingan amino acid sequence of KASQDVSIAVA; CDR2 comprising an amino acidsequence of SASYRYT; and CDR3 comprising an amino acid sequence ofQQHYITPLT; and the CDRs of the heavy chain variable region of thehumanized RS7 MAb comprises CDR1 comprising an amino acid sequence ofNYGMN; CDR2 comprising an amino acid sequence of WINTYTGEPTYTDDFKG andCDR3 comprising an amino acid sequence of GGFGSSYWYFDV.
 8. The humanizedantibody or fragment thereof of claim 7, further comprising the FRs ofthe light and heavy chain constant regions of a human antibody.
 9. Thehumanized antibody or fragment thereof of claim 3, wherein the FRs ofthe light or heavy chain variable regions of said humanized antibodycomprise at least one amino acid substituted by an amino acid residuefound at a corresponding location in the murine RS7 MAb.
 10. Thehumanized antibody or fragment thereof of claim 9, wherein at least oneof the substituted amino acid residues is at the location selected fromthe group consisting of amino acid residue 38, 46, 68 and 91 of themurine heavy chain variable region of FIG. 3B.
 11. The humanizedantibody or fragment thereof of claim 9, wherein at least one of thesubstituted amino acid residues is at the location selected from thegroup consisting of amino acid residue 20, 85 and 100 of the murinelight chain variable region FIG. 3A.
 12. The antibody or fragmentthereof of claim 2, wherein said antibody or fragment thereof ischimeric.
 13. The antibody or fragment thereof of claim 12, wherein saidantibody or fragment thereof comprises RS7 V_(K) nucleotide sequence ofFIG. 2A and the RS7 V_(H) nucleotide sequence of FIG. 2B.
 14. Theantibody or fragment thereof of claim 3, wherein said antibody orfragment thereof comprises a hRS7 V_(K) nucleotide sequence of FIG. 4Aand a hRS7 V_(H) nucleotide sequence of FIG. 4B.
 15. A cancer celltargeting diagnostic or therapeutic conjugate comprising an antibodycomponent comprising an anti-EGP-1 MAb or fragment thereof or anantibody fusion protein or fragment thereof of claims 1, that binds tosaid cell, wherein said antibody component is bound to at least onediagnostic or at least one therapeutic agent.
 16. A multivalent,multispecific antibody comprising one or more antigen-binding siteshaving affinity toward a EGP-1 target antigen and one or more haptenbinding sites having an affinity toward a hapten molecule.
 17. Anantibody fusion protein or fragment thereof comprising at least twoanti-EGP-1 MAbs or fragments thereof, wherein said MAbs or fragmentsthereof are selected from said MAb or fragment thereof claim
 1. 18. Anantibody fusion protein or fragment thereof comprising at least onefirst anti-EGP-1 MAb or fragment thereof of claim 1, and at least onesecond MAb or fragment thereof, wherein said second MAb is acarcinoma-associated antibody.
 19. A DNA sequence comprising a nucleicacid encoding a MAb or fragment thereof selected from the groupconsisting (a) an anti-EGP-1 MAb or fragment thereof of claim 1; (b) anantibody fusion protein or fragment thereof comprising at least two ofsaid MAbs or fragments thereof, (c) an antibody fusion protein orfragment thereof comprising at least one first anti-EGP-1 MAb orfragment thereof comprising said MAb or fragment thereof of claim 1 andat least one second MAb or fragment thereof, other than the MAb orfragment thereof of claim 1; and (d) an antibody fusion protein orfragment thereof comprising at least one first MAb or fragment thereofcomprising said MAb or fragment thereof of claim 1 and at least onesecond MAb or fragment thereof, wherein said second MAb is an antibodyor fragment reactive with the antigen selected from the group consistingof CEA, CSAp, Tn, Le(y), MUC-1-4, Tag-72, EGFR, HER2/neu, PSMA, PSA,AFP, HCG, HCG-beta, ferritin, PAP, PLAP, EGP-2, histone, cytokeratin,Tenascin, CanAg, kidney cancer G 250, VGFR1, VGFR2, VEGF, P1GF,insulin-like growth factor, oncogene products, and a combinationthereof.
 20. An expression vector comprising the DNA sequence of claim19.
 21. A host cell comprising the DNA sequence of claim
 19. 22. Amethod of delivering a diagnostic or therapeutic agent, or a combinationthereof, to a target comprising (i) providing a composition thatcomprises an anti-EGP-1 antibody bound to at least one diagnostic or atleast one therapeutic agent and (ii) administering to a subject in needthereof said composition.
 23. A method of delivering a diagnostic agent,a therapeutic agent, or a combination thereof to a target, comprising:(i) administering to a subject the antibody of claim 16; (ii) waiting asufficient amount of time for an amount of the non-binding antibody toclear a subject's blood stream, and (iii) administering to said subjecta hapten comprising a diagnostic agent, a therapeutic agent, or acombination thereof, that binds to a binding site of said antibody. 24.The method of claim 23, wherein said hapten binds to more than onebinding site of said antibody.
 25. A method for diagnosing or treatingcancer, comprising: (i). administering to a subject in need thereof theantibody of claim 16; (ii). waiting a sufficient amount of time for anamount of the non-binding protein to clear the subject's blood stream;and (iii). administering to said subject a hapten comprising adiagnostic agent, a therapeutic agent, or a combination thereof, thatbinds to a binding site of said antibody.
 26. A method of treating amalignancy in a subject comprising administering to said subject atherapeutically effective amount of an antibody fusion protein orfragment thereof comprising at least two MAbs or fragments thereof,wherein said MAbs are selected from said MAbs of claim 1, or comprisingat least one MAb or fragment thereof of claim 1 formulated in apharmaceutically suitable excipient.
 27. A method of treating amalignancy in a subject comprising administering to said subject atherapeutically effective amount of a therapeutic conjugate comprising aEGP-1 MAb or fragment thereof or an antibody fusion protein or fragmentthereof of claim 1, wherein said EGP-1 MAb or fragment thereof orantibody fusion protein or fragment thereof is bound to at least onetherapeutic agent, formulated in a pharmaceutically suitable excipient.28. The method of claim 27, wherein a first binding site of theanti-EGP-1 antibody or fragment thereof is present in a multivalent,multispecific fusion protein or chemical conjugate and a second bindingsite is reactive with a tumor marker substance other than EGP-1.
 29. Themethod of claim 27, further comprising a second antibody or fragmentthereof or antibody fusion protein or fragment thereof
 30. The method ofclaim 29, wherein said second antibody or fragment thereof is conjugatedto at least one therapeutic or diagnostic agent.
 31. A method ofdiagnosing a malignancy in a subject comprising administering to saidsubject a therapeutically effective amount of a therapeutic conjugatecomprising a anti-EGP-1 MAb or fragment thereof or an antibody fusionprotein or fragment thereof of claim 1, wherein said anti-EGP-1 MAb orfragment thereof or antibody fusion protein or fragment thereof is boundto at least one diagnostic agent, formulated in a pharmaceuticallysuitable excipient.
 32. A method of treating a cancer cell in a subjectcomprising (i) administering to said subject a therapeutically effectiveamount of a composition comprising a naked anti-EGP-1 MAb or fragmentthereof or a naked antibody fusion protein or fragment thereof of claim1, (ii) formulating said naked EGP-1 MAb or fragment thereof or antibodyfusion protein or fragment thereof in a pharmaceutically suitableexcipient.
 33. The method of claim 32, wherein said composition furthercomprises a second antibody or fragment thereof not in claim
 1. 34. Themethod of claim 33, wherein said naked anti-EGP-1 antibody or fragmentthereof or naked antibody fusion protein or fragment thereof isadministered before, in conjunction with, or after a second nakedantibody or fragment thereof reactive with a second tumor markerexpressed by said malignancy is administered to said subject.
 35. Themethod of claim 32, wherein said naked anti-EGP-1 antibody isadministered before, concurrently or after a therapeutic agent.
 36. Amethod of diagnosing a malignancy in a subject comprising (i) performingan in vitro diagnosis assay on a specimen from said subject with acomposition comprising a naked anti-EGP-1 MAb or fragment thereof or anaked antibody fusion protein or fragment thereof of claim 1.